206?The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 117? ? ? Number 1? ? ? January 2007
The type III TGF-β receptor suppresses
breast cancer progression
Mei Dong,1 Tam How,1 Kellye C. Kirkbride,1,2 Kelly J. Gordon,1,2 Jason D. Lee,1,2 Nadine Hempel,1
Patrick Kelly,2 Benjamin J. Moeller,3 Jeffrey R. Marks,4 and Gerard C. Blobe1,2
1Department of Medicine, 2Department of Pharmacology and Cancer Biology, 3Department of Radiation Oncology, and
4Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA.
TGF-β is a member of a superfamily of functionally diverse, but
structurally conserved, cytokines that regulate cell proliferation,
differentiation, apoptosis, and motility in a cell- and context-spe-
cific manner (1). TGF-β exerts these biological effects by binding
to 2 high-affinity cell surface receptors, the type II TGF-β receptor
(TβRII) and the type III TGF-β receptor (TβRIII, or betaglycan);
TβRIII functions as a coreceptor to increase ligand binding to
TβRII. Once bound to TGF-β, TβRII recruits, binds, and trans-
phosphorylates the type I TGF-β receptor (TβRI), thereby stimu-
lating its protein kinase activity. The activated TβRI phosphory-
lates transcription factors Smad2 or Smad3, which then binds to
Smad4. The resulting Smad complex translocates into the nucleus
and interacts with other transcription factors to specifically regu-
late the transcription of a multitude of TGF-β–responsive genes.
TGF-β has an important role in normal mammary biology as a
potent inhibitor of mammary epithelial proliferation and regula-
tor of mammary ductal and alveolar development (2, 3). Early in
mammary carcinogenesis the TGF-β signaling pathway functions
as a tumor suppressor, with most human breast cancers developing
resistance to the growth-inhibitory effects of TGF-β and with elevat-
ed levels of TGF-β associated with decreased incidence of mammary
cancer in mouse models (4) and decreased breast cancer incidence in
humans (5, 6). However, at later stages of mammary carcinogenesis,
levels of TGF-β increase with tumor progression (7–9) and confer a
poorer prognosis for human breast cancer patients (10).
Although the TGF-β signaling pathway has an important role in
regulating mammary carcinogenesis, alterations in the main com-
ponents of the pathway, including TβRII, TβRI, Smad2, Smad3,
and Smad4, are infrequent in human breast cancers (6, 11). A role
for the TGF-β coreceptor TβRIII as a mediator and regulator of
TGF-β signaling has emerged as a result of recent studies, with
essential roles in chick heart and mouse development (12, 13) and
in regulating TβRII and TβRI cell surface expression and inter-
nalization as well as TGF-β signaling (14, 15). TβRIII has been
reported to be expressed at low levels in the MCF-7 human breast
cancer cell line (16), and restoring TβRIII expression in these cells
suppresses their anchorage-independent growth in vitro as assayed
by colony formation in soft agarose (16), while increasing TβRIII
expression in MDA-MB231 breast cancer cells suppresses their
tumorigenicity in vivo as assessed by tumor formation in athymic
nude mice (17). These results suggest that decreased TβRIII expres-
sion may be a mechanism for altering TGF-β responsiveness dur-
ing mammary carcinogenesis. Here we demonstrate that TβRIII is
a suppressor of breast cancer progression and that, when TβRIII
expression is restored in human breast cancer cells, breast tumor
invasion, angiogenesis, and metastasis are inhibited in vivo.
Decreased TβRIII expression in human breast cancer. As evidence sup-
porting roles for TβRIII in regulating TGF-β signaling have emerged
(12–15), and a low level of TβRIII expression has been reported in
the MCF-7 human breast cancer cell line (16), we investigated the
expression status of TβRIII in human breast cancer. Breast cancers
are classified into different histologic subtypes, with invasive duc-
tal carcinoma (IDC) being the most common (~70%), followed by
lobular carcinoma (~8%). The development of IDC has been pro-
posed to follow a stepwise process — including ductal carcinoma
in situ (DCIS) — culminating in the potentially lethal stage of IDC.
We initially analyzed a cDNA array containing 50 human breast
cancer samples with matched normal controls (Figure 1A). TβRIII
mRNA levels were reduced in 60% of the lymph node–negative IDCs
(2.64 ± 0.49–fold), 64.7% of the lymph node–positive IDCs (2.47 ± 0.29–
fold) and in 100% of the IDCs with distant metastasis (3.98 ± 0.79–
Nonstandard?abbreviations?used: DCIS, ductal carcinoma in situ; ER, estrogen
receptor; IDC, invasive ductal carcinoma; IHC, immunohistochemical; LOH, loss
of heterozygosity; PCNA, proliferating cell nuclear antigen; sTβRIII, soluble TβRIII;
TβRI, type I TGF-β receptor; TβRII, type II TGF-β receptor; TβRIII, type III TGF-β
receptor; 4T1-Neo cells, 4T1 cells stably expressing the pcDNA-Neo expression vector;
4T1-TβRIII cells, 4T1 cells stably expressing TβRIII.
Conflict?of?interest: The authors have declared that no conflict of interest exists.
Citation?for?this?article: J. Clin. Invest. 117:206–217 (2007). doi:10.1172/JCI29293.
?The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 117? ? ? Number 1? ? ? January 2007
fold) as well as in all histological subtypes represented in Figure 1B,
suggesting an increased frequency of loss with disease progression.
TβRIII mRNA levels were also significantly reduced in 83.3% of
lobular carcinomas (2.84 ± 0.40–fold). We also examined 3 sets of
specimens on the cDNA array with matched normal breast, prima-
ry breast cancer, and metastatic breast cancer tissue from the same
patient. In all 3 cases, TβRIII expression decreased from normal
breast to primary breast cancer to metastatic breast cancer, with an
average 88% decrease in expression from normal breast to primary
breast cancer and a further 61% decrease from primary breast cancer
to metastatic breast cancer (Figure 1C; P < 0.0001), suggesting pro-
gressive loss of TβRIII expression with cancer progression.
To confirm decreased expression of TβRIII and establish
its association with breast cancer progression, we performed
immunohistochemical (IHC) analysis for TβRIII expression on a
breast cancer tissue array containing 252 breast cancers of different
stages (20 DCIS, 64 lymph node–negative, 64 lymph node–positive,
and 64 distant metastatic) and 40 normal breast specimens with
available pathologic information including tumor size, TNM stage,
number of nodes positive, invasive grade, and estrogen receptor
(ER) and progesterone receptor status. TβRIII expression progres-
sively decreased from normal breast specimens (Figure 2, A and B)
to DCIS to lymph node–negative breast cancer. The proportion with
abundant TβRIII expression decreased from 68.4% in normal breast
specimens to 23.5% in DCIS specimens to 5.1% in lymph node–nega-
tive breast cancer specimens (P < 0.01, 2-tailed Fisher’s exact prob-
ability). At the same time, the proportion with no TβRIII expression
increased from 0% in normal breast specimens to 23.5% in DCIS
specimens to 67.8% in lymph node–negative breast cancer specimens
(P < 0.01, 2-tailed Fisher’s exact probability). In DCIS specimens
with loss of TβRIII expression (Figure 2A, arrow), TβRIII was pres-
ent in adjacent normal-appearing breast ducts (Figure 2A, arrow-
head), which served as a useful internal control. To directly address
the role of loss of TβRIII expression in breast cancer progression,
we assessed matched tissue sets for which either matching normal
breast and invasive breast cancer specimens (Figure 2C) or matching
DCIS and invasive breast cancer specimens (Figure 2D) were avail-
able for analysis. In addition, one of these samples had matching
normal breast, DCIS, and invasive breast cancer specimens available
for analysis. When examining TβRIII expression in matched nor-
mal breast and invasive breast cancer specimens, TβRIII expression
decreased in every case (10 of 10), with 6 cases decreasing from high
expression (IHC score of 5) in normal breast tissue to low expres-
sion (IHC score of 0–1) in the matching invasive breast cancer tissue
(Figure 2C). When examining TβRIII expression in matched DCIS
and invasive breast cancer specimens, TβRIII expression decreased
in 63% of the cases (5 of 8), with 1 additional case where expression
was already absent at the DCIS stage (Figure 2D). In the sample with
matching normal breast, DCIS, and invasive breast cancer speci-
mens, TβRIII expression decreased from an IHC score of 5 in the
Loss of TβRIII mRNA expression during mammary carcinogenesis. (A) TβRIII mRNA levels were detected by hybridizing [32P]-labeled human
TβRIII cDNA probe to the Clontech Cancer Profiling Array I. The portion of the array containing breast samples is shown, with tumor specimens
(T) and matched normal breast tissue (N). Asterisks indicate metastatic specimens corresponding to the normal and tumor samples spotted on
the immediate left. (B) Quantitative data were obtained by analyzing the array with NIH ImageJ software, summarized as the ratio relative to
normal breast, and expressed as mean ± SEM. (C) Quantitative data from matched normal, primary breast tumor, and metastatic breast tumor
tissue expressed as mean ± SEM. ***P < 0.0001, ANOVA.
208? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 117? ? ? Number 1? ? ? January 2007
normal breast specimen to 2 in the DCIS specimen to 0 in the inva-
sive breast cancer specimen. These data indicate that TβRIII expres-
sion is significantly decreased in breast cancer, with loss of TβRIII
expression correlating with breast cancer progression.
Loss of heterozygosity and transcriptional downregulation of the TβRIII
gene in human breast cancer. Members of the TGF-β signaling path-
way, including TβRII and Smad4, frequently have inactivating
mutations in human cancers (18, 19). To investigate whether there
are mutations in the TβRIII gene, TGFBR3 (216 kb of genomic
DNA composed of 17 exons), that could abrogate TβRIII function
in breast cancer, sequence analysis of the 16 coding exons (exon 1
is untranslated) was carried out on 20 primary breast cancer DNA
samples. Although several polymorphisms were detected (data not
shown), no mutations were found. Thus, TGFBR3 does not appear
to be a target for mutational inactivation in breast cancer.
TGFBR3 maps to chromosome 1p32, a region that has been report-
ed to exhibit loss of heterozygosity (LOH) in a variety of human
cancers, including breast cancer (20–22). Therefore, to investigate
the mechanism for loss of TβRIII expression during breast tumori-
genesis, we examined LOH at the TGFBR3 locus using microsatellite
markers on DNA samples extracted from 26 human breast cancer
specimens and the matching normal peripheral lymphocytes. With
4 microsatellite markers immediately adjacent to and within the
TGFBR3 locus, we were able to establish that 50% (13 of 26) of these
samples exhibited LOH at the TGFBR3 locus (Figure 3, A and B),
closely matching the 43%–61% LOH reported for the 1p region and
the 58% reported for 1p32 in human breast cancers (20–22). LOH
at the TGFBR3 locus correlated with loss of TβRIII expression, with
75% (9 of 12) of those with the lowest TβRIII expression exhibiting
LOH at the TGFBR3 locus and only 20% (1 of 5) with the highest
TβRIII expression exhibiting LOH at the TGFBR3 locus (Figure 3C).
Taken together, these data support LOH as a mechanism for loss of
TβRIII expression in breast cancer.
During later stages of mammary carcinogenesis, levels of TGF-β
increase with tumor progression (7–9) and confer a poorer progno-
sis for human breast cancer patients (10). As TGF-β isoforms have
previously been demonstrated to decrease TβRIII promoter activity
(23), we assessed whether the elevated levels of TGF-β could repress
TβRIII expression at the transcriptional level in breast cancer cells.
In MDA-MB231 breast cancer cells, which exhibit basal TβRIII
expression, TGF-β1 treatment resulted in a significant (up to 80%)
reduction in the TβRIII mRNA level (Figure 3D). This effect was rel-
atively specific for TβRIII, as TGF-β1 treatment slightly increased
TβRI mRNA levels and decreased TβRII mRNA levels by less than
50% (Figure 3D). These results suggest that, apart from LOH, tran-
scriptional downregulation due to increased TGF-β in the breast
Progressive loss of TβRIII protein expression during mammary carcinogenesis. (A) Representative IHC analysis of TβRIII expression (original
magnification, ×40) in normal breast ductal cells, in different grades of DCIS, and in lymph node–negative (node neg) and –positive (node pos)
IDC. Insets depict staining of entire tissue core (original magnification, ×10). Immunoreactivity for TβRIII was scored as 0–5 and categorized as
low (0–1), medium (2–3), or high (4–5). Note the absence of TβRIII staining in IDC and high-grade DCIS (arrows) versus presence of staining in
normal ducts and normal-appearing ducts adjacent to the DCIS lesion (arrowhead). (B) Summary of IHC results, with percentages shown. Dis
met, distant metastasis. **P < 0.01, 2-tailed Fisher’s exact probability. (C) Patient-matched normal and invasive breast cancer IHC TβRIII scores.
(D) Patient-matched DCIS and invasive breast cancer IHC TβRIII scores.
?The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 117? ? ? Number 1? ? ? January 2007
cancer microenvironment could be another mechanism leading to
decreased TβRIII expression during mammary carcinogenesis.
TβRIII delays and decreases metastatic potential of breast cancer cells in vivo.
The frequent loss of TβRIII expression observed during progression
to invasive disease suggested that TβRIII loss during mammary car-
cinogenesis may specifically promote tumor invasion and metastasis
in vivo. To investigate a causal role for decreased TβRIII expression
in breast cancer progression, we examined the effect of TβRIII on in
vivo tumor growth and metastasis using a murine model for mam-
mary carcinogenesis. Murine 4T1 mammary cancer cells, which are
derived from a BALB/c murine mammary tumor, share many char-
acteristics with human mammary cancers including spontaneous
lung metastasis in immunocompetent mice and have been widely
used as a model of breast cancer (24, 25). The 4T1 cells were geneti-
cally engineered to express the firefly luciferase gene so that by peri-
odically injecting the substrate luciferin into mice carrying these cells
and taking bioluminescent images, we were able to closely and quan-
titatively follow their in vivo growth and metastatic potential. The
4T1 cells were stably transfected with TβRIII (4T1-TβRIII cells, see
Supplemental Figure 1; supplemental material available online with
this article; doi:10.1172/JCI29293DS1), resulting in 4T1 cells with
increased TβRIII expression. The 4T1-TβRIII cells and control 4T1
cells stably expressing the pcDNA-Neo expression vector (4T1-Neo
cells) were injected into the axillary mammary fat pads of BALB/c
mice. The primary tumor was measured every 2 days starting from
day 10 after injection and removed on day 20. Tumor metastases were
then followed by bioluminescent imaging every 3 days over a period
of 19 days. No significant difference was observed in the growth of
the primary tumors from 4T1-TβRIII and 4T1-Neo cells as shown by
the growth curve (Figure 4A) and tumor mass at the time of resection
(Figure 4B), establishing that TβRIII had no effect on tumorigenicity
in vivo. However, mice injected with 4T1-TβRIII cells demonstrated
a significantly delayed onset of tumor metastasis as well as a signifi-
cant reduction in both the size and number of lung metastases com-
pared with the mice injected with control 4T1-Neo cells (Figure 4,
C–E). In addition, while no tumor recurrence at the primary site or
animal death was observed in mice injected with 4T1-TβRIII cells, the
control mice with the 4T1-Neo cells had a 20% local recurrence rate
and a 13.3% death rate during the study (Table 1).
Further pathologic examination of the primary tumors dem-
onstrated that the 4T1-Neo tumors exhibited increased invasion
of the surrounding normal mammary tissue (Figure 5A) and skin
(Figure 5B), while the 4T1-TβRIII tumors exhibited little to no
invasion and instead maintained a distinct margin with the adja-
cent normal tissue (Figure 5C). In addition, primary recurrences in
the 4T1-Neo mice exhibited invasion of tumor cells into the blood
vessels, resulting in internal hemorrhage (Figure 5D). Pathologic
examination of tumor metastasis revealed distant metastasis to the
Frequent LOH of the TGFBR3
gene locus in human breast can-
cers correlates with loss of TβRIII
mRNA expression. LOH analysis
was performed on DNA extract-
ed from 26 human breast cancer
specimens and matching normal
lymphocytes. (A) Representa-
tive results showing allelic loss in
tumors 1, 2, and 6 (denoted by
asterisks) when PCR products
were separated on a MetaPhor
agarose gel. Microsatellite mark-
ers D1S1588 and D1S188 are
described in Methods. (B) LOH
was confirmed using an ABI
sequencer and quantified using
GeneScan software. A represen-
tative sample with LOH is shown.
(C) Quantitative real-time PCR
analysis of TβRIII mRNA levels
in breast cancer specimens with
(red bars) and without (black
bars) LOH. (D) Quantitative real-
time PCR analysis of mRNA lev-
els of TβRI, TβRII, and TβRIII in
MDA-MB231 cells in response to
TGF-β1 (100 pM) stimulation for
the indicated times.
210? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 117? ? ? Number 1? ? ? January 2007
mesentery (Figure 5E), the paratracheal lymph nodes (Figure 5F),
and the cecum in addition to the lung in control 4T1-Neo mice,
while 4T1-TβRIII exhibited only lung metastases. In addition,
when lung metastases were observed in 4T1-TβRIII mice, these
TβRIII delayed and decreased metastatic potential of breast cancer cells in vivo. Either 4T1-Neo (Neo) or 4T1-TβRIII (RIII) cells (75,000 cells/
mouse) were implanted into the axillary mammary fat pads of BALB/c mice. (A) Primary tumor growth was recorded by measuring tumor size
every 2 days beginning at 10 days after injection and presented as mean ± SEM. (B) Weight of the primary tumors upon surgical removal on
day 20 after injection. Data are mean ± SEM (n = 16). (C) Bioluminescence imaging was performed every 3 postoperative days (POD). Repre-
sentative images are shown. Red and violet signals correspond to the maximum and minimum intensity values, respectively, with other colors
representing the values in between. (D) Record of luminescent signals for every mouse in each group at the indicated time points. (E) Average
luminescent signal in each group at the indicated time points. **P < 0.01.
? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 117? ? ? Number 1? ? ? January 2007
metastatic lesions were always small, well circumscribed, and iso-
lated (Figure 5, H and I) compared with the large, locally invasive
lung metastases observed in 4T1-Neo mice (Figure 5G). These
studies support a specific suppressor effect of TβRIII on cellular
invasiveness and metastasis, but not on primary tumorigenesis.
TβRIII decreases angiogenesis in vivo. Cancer metastasis is a multi-
step process requiring the cells growing at the primary site to invade
through the basement membrane, enter lymph or blood vessels,
extravasate from the vessel, and then grow at the distant site. Many of
the processes involved in primary tumorigenesis and growth
of metastases are similar, including increased proliferation,
decreased apoptosis, and increased angiogenesis. To further
establish the mechanism of TβRIII on decreasing metastasis in
vivo, we performed immunohistochemistry for the prolifera-
tion marker proliferating cell nuclear antigen (PCNA), TUNEL
staining as a marker for apoptosis, and immunohistochemistry
for CD31 as an endothelial surface marker on primary tumors
and metastatic lesions. There were no significant differences
observed in PCNA or TUNEL staining in 4T1-Neo and 4T1-
TβRIII primary tumors or lung metastases (Figure 6A), sug-
gesting that differences in proliferation or apoptosis did not
account for the differential metastatic behavior of 4T1-Neo
and 4T1-TβRIII cells. However, CD31 staining revealed a
decrease in the number of tumor-associated blood vessels per
field, smaller vessel diameters, and less staining intensity in
4T1-TβRIII tumors (Figure 6B), which supported an inhibitory
effect of TβRIII on tumor angiogenesis. Taken together, these
data indicate that loss of TβRIII expression facilitates tumor
metastasis in vivo not only through an increase in tumor cell
invasiveness but also through enhanced tumor angiogenesis.
TβRIII inhibits the invasiveness of breast cancer cells through
the generation of soluble TβRIII. To further define the mecha-
nisms by which TβRIII regulated breast cancer invasiveness
and metastasis in vivo, we examined the effect of increasing
TβRIII expression on the invasiveness of breast cancer cell
lines in vitro. We initially assessed the 4T1-Neo and 4T1-
TβRIII cell lines; however, these cell lines both tended to
aggregate and were not significantly invasive in vitro (data
not shown). Therefore, we used the tumorigenic, invasive,
and metastatic MDA-MB231 cell line. Overexpression of
TβRIII had no significant effect on the rate of cell division,
nor did it restore cell responsiveness to TGF-β–induced
growth inhibition (Supplemental Figure 2). However, it
dramatically repressed the ability of MDA-MB231 cells to
invade through Matrigel and significantly attenuated the
responsiveness of the MDA-MB231 cells to TGF-β–induced
invasion (Figure 7, A–C). These results confirm a direct effect of
TβRIII on inhibiting breast cancer cell invasiveness.
We next assessed the ability of specific TβRIII mutants to mediate
this function. Interestingly, a TβRIII mutant lacking the entire cyto-
plasmic domain inhibited breast cancer cell invasiveness to an extent
similar to that of full-length TβRIII (Figure 7, A–C), suggesting that
the effect of TβRIII on regulating invasion is independent of func-
tions mediated by the cytoplasmic domain of TβRIII, including bind-
ing Gα-interacting protein–interacting protein, C terminus (GIPC)
(26) and β-arrestin2 (15) and mediating TGF-β signaling (14).
The extracellular domain of TβRIII can be proteolytically cleaved
in the juxtamembrane region (27), and the resulting soluble TβRIII
(sTβRIII) has been demonstrated to suppress tumor growth and
angiogenesis, potentially through binding and sequestering TGF-β
and preventing signaling through the membrane-bound receptors
(28). To assess whether the effects of TβRIII could be mediated
by the production of sTβRIII, we first examined whether the 4T1-
TβRIII and MDA-MB231–TβRIII cells lines produced sTβRIII.
We collected conditioned media from each cell line, crosslinked
iodinated TGF-β1, and specifically immunoprecipitated sTβRIII
with an antibody to the extracellular domain. These studies con-
firmed that both the 4T1-TβRIII and the MDA-MB231–TβRIII
cell lines produced a significant amount of sTβRIII (Figure 7F).
TβRIII decreases metastasis in vivo
Local recurrence rate
Distant metastatic sites
Average tumor load
on POD19 (photons/s)
Death rate by POD19
20% (3 of 15)
Lung, cecum, mesentery
80% (12 of 15)
1.12 × 108
53.3% (8 of 15)
3.7 × 107
13.3% (2 of 15) 0%
POD, postoperative day.
TβRIII decreased tumor cell invasiveness and metastasis in vivo. Repre-
sentative H&E staining (original magnification, ×10) of (A and B) primary
tumors from mice implanted with 4T1-Neo cells exhibiting local invasion (red
arrows) of tumor cells into the adjacent normal mammary tissue (A) and skin
(B); (C) a representative primary tumor from mice implanted with 4T1-TβRIII
cells demonstrating the absence of local invasion, as indicated by the clear
margin between the tumor and the adjacent normal mammary tissue (yellow
arrow); (D) a recurring tumor in a mouse at the primary injection site of 4T1-
Neo cells exhibiting internal bleeding due to invasion of tumor cells into the
blood vessels; (E) a metastatic tumor (black arrow) adjacent to the pancreas
(green arrowhead) found on the mesentery of a mouse implanted with 4T1-
Neo cells; (F) a significantly enlarged paratracheal lymph node adjacent to
the trachea (blue arrowhead) containing metastatic tumor cells (black arrow)
in a mouse with 4T1-Neo cells, indicating the presence of lymphatic metas-
tasis; (G) multiple large metastatic tumor nodules (black arrows) in the lung
of a mouse implanted with 4T1-Neo cells; and (H and I) representative lung
metastases in mice implanted with 4T1-TβRIII cells (black arrows).
212?The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 117? ? ? Number 1? ? ? January 2007
Accordingly, we examined the effect of sTβRIII on MDA-MB231
breast cancer cell invasion in vitro. Conditioned media collected
from COS-7 cells transiently transfected with full-length TβRIII
or sTβRIII potently decreased TGF-β–induced invasion of MDA-
MB231 breast cancer cells through Matrigel (Figure 7, D and E).
As sTβRIII mediated the effects of TβRIII expression on breast
cancer invasiveness in vitro and in vivo, we reasoned that TβRIII
would attenuate TGF-β signaling in the MDA-MB231–TβRIII cells
in vitro and in the 4T1-TβRIII tumors in vivo. To examine the effect
of TβRIII expression on activation of the Smad pathway in response
to TGF-β stimulation, MDA-MB231–TβRIII and MDA-MB231–
Neo breast cancer cells were treated with TGF-β, and phosphory-
lation levels of Smad2 were quantified. As shown in Figure 8A,
TβRIII expression in the MDA-MB231 cells resulted in reduced
TGF-β–stimulated Smad2 phosphorylation compared with the
MDA-MB231–Neo cells. In addition, TGF-β1–mediated activa-
tion of TGF-β1–responsive, Smad-dependent promoter pE2.1
was also reduced in the MDA-MB231–TβRIII cells (Figure 8B).
Consistent with this in vitro result, immunohistochemistry of the
mouse mammary tumors revealed decreased frequency and inten-
sity of phosphorylated Smad2 nuclear staining in the 4T1-TβRIII
tumors compared with the 4T1-Neo tumors (Figure 8C). Further
support for a significant role for sTβRIII in mediating the effects
of TβRIII was provided by the decreased angiogenesis demonstrat-
ed in the 4T1-TβRIII tumors in vivo (Figure 6B), as sTβRIII has
been demonstrated to decrease angiogenesis in vivo (28, 29).
sTβRIII is produced from cells and tissues from 7 different
mammalian species, including humans (30, 31), and has also been
detected in serum (30) and human milk (32). In addition, the
expression of sTβRIII has been demonstrated to closely correlate
with the cell surface expression of TβRIII (30), suggesting that it is
released constitutively. To support a physiological role for sTβRIII
in mediating the effects of TβRIII on breast cancer invasiveness, we
examined expression of sTβRIII in a panel of human breast epithe-
lial and breast cancer cell lines. sTβRIII was expressed in all human
breast cell lines tested, including the human mammary epithelial
cell line MCF-10A and the human breast cancer cell lines MCF-7,
T47D, and MDA-MB231 (Supplemental Figure 3A). As previously
reported, the level of sTβRIII usually correlated with cell surface
expression of TβRIII. Finally, we examined expression of sTβRIII
in plasma from normal human volunteers as well as from patients
with breast cancer. While we detected expression of sTβRIII (a het-
TβRIII inhibits tumor angiogenesis without altering cancer cell proliferation and apoptosis in vivo. (A) Tissue sections of primary tumors and lung
metastases from mice implanted with 4T1-Neo and 4T1-TβRIII cells were immunostained for PCNA and TUNEL to evaluate cell proliferation
and apoptosis, respectively. Representative staining frequency and intensity is shown (original magnification, ×40). (B) Immunostaining of CD31
(original magnification, ×10) was performed as a marker to evaluate angiogenesis. Note the decreased number and size of tumor-associated
blood vessels as well as decreased staining intensity (insets; original magnification, ×100) in 4T1-TβRIII primary tumors and lung metastases.
Values are the averages from 6 mice and expressed as mean ± SD. *P < 0.05; **P < 0.01.
?The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 117? ? ? Number 1? ? ? January 2007
erogeneous product from approximately 65–250 kDa) in plasma
in all (5 of 5) of the normal human volunteers, we did not detect
sTβRIII in the plasma of any breast cancer patients (0 of 13; Sup-
plemental Figure 3B). Taken together, these data support a model
in which ectodomain shedding of TβRIII produces sTβRIII, which
then functions to attenuate TGF-β–mediated invasiveness of breast
cancer cells and tumor-induced angiogenesis in vitro and in vivo.
Decreased TβRIII expression correlates with decreased recurrence-free
survival in breast cancer patients. As decreased TβRIII expression is
frequently observed in human breast cancers and restoring TβRIII
expression decreased invasiveness and metastasis in vivo, we explored
whether TβRIII expression could be a useful prognostic marker for
breast cancer patients. We examined publicly available microarray
data sets in which both TβRIII expression and recurrence-free
survival data were available (33–36). We set TβRIII expression as a
dichotomous variable, with high expression as above the mean and
low expression as below the mean. In the largest data set (that of
Wang et al., ref. 36), composed of 286 patients with lymph node–
negative breast cancers, low expression of TβRIII was significantly
associated with a decrease in recurrence-free survival (Figure 9;
P = 0.043), with recurrence defined as a distant metastatic event.
The hazard ratio (HR) for recurrence based on TβRIII expression
(HR, 1.569) was higher than that for ER status (HR, 1.18) or for
Her2/Neu status (HR, 1.06) (37). In addition, we examined wheth-
er the predictive value of TβRIII was independent of other known
prognostic factors. As all samples in the Wang et al. data set (36)
came from lymph node–negative patients, we analyzed the only
other available prognostic factor within the data set, ER status. A
Pearson correlation coefficient of –0.08 (95% confidence interval,
–0.19 to 0.036) supported little correlation between TβRIII expres-
sion and ER status, although the data set was not large enough to
power the analysis (P = 0.177). In 3 other completely independent
data sets (Sorlie et al., ref. 34, containing 74 locally advanced ER-
positive and -negative primary breast cancers; van’t Veer et al., ref.
33, containing 97 ER-positive and -negative lymph node–negative
breast cancers; and Ma et al., ref. 35, containing 60 hormone recep-
tor–positive breast cancers), there was a trend toward decreased
recurrence-free survival associating with low TβRIII expression,
although in each case the number of patients was not large enough
to reach statistical significance (data not shown). Taken together,
these data suggest that TβRIII expression is predictive of recur-
rence-free survival in breast cancer patients.
Restoration of TβRIII expression inhibits Matrigel invasiveness of MDA-MB231 breast cancer cells. (A) MDA-MB231 cells were infected with
equivalent amounts of adenoviral constructs carrying GFP, HA-tagged TβRIII, and a TβRIII mutant lacking the entire cytoplasmic domain
(TβRIIIΔcyto). Expression of the transgenes was confirmed by Western blotting of cell lysate using anti-HA antibody. (B and C) Matrigel invasion
assay. Adenovirally infected MDA-MB231 cells (75,000 cells) were seeded in a Matrigel-coated upper chamber and treated with TGF-β1 (15 pM)
2 hours later. Cell invasion through the Matrigel after 24 hours’ incubation was detected by H&E staining and quantitated. (D and E) Matrigel
invasion assay was performed after resuspending MDA-MB231 cells in the conditioned media collected from pcDNA3.1-Neo–, TβRIII-, and
sTβRIII-transfected COS-7 cells. Data are mean ± SEM, n = 3 in triplicate. **P < 0.01. (F) Detection of sTβRIII in media of MDA-MB231–TβRIII
and 4T1-TβRIII cells by [125I]TGF-β1–binding crosslinking followed by immunoprecipitation.
214? The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 117? ? ? Number 1? ? ? January 2007
Breast cancer is the leading cause of cancer death in women in the
world, with most breast cancer morbidity and mortality resulting
from metastatic disease (38). Although the TGF-β signaling path-
way has an important role in mammary carcinogenesis, the major
components of the pathway, including the signaling receptors, TβRII
and TβRI, and the predominant signaling pathway downstream of
these receptors, Smad2, Smad3, and Smad4, are usually intact in
human breast cancers (6, 11). In the present study, we demonstrate
that expression of the TGF-β coreceptor TβRIII was frequently
decreased at the mRNA and protein levels in human breast cancer,
with approximately 90% of specimens demonstrating decrease or loss
at the mRNA level and approximately 70% demonstrating decrease
or loss at the protein level. Thus, we believe loss of TβRIII expression
to be the most common alteration in the TGF-β signaling pathway
described in human breast cancer to date. We have further demon-
strated that loss of TβRIII expression was an early event, occurring
initially in the preinvasive state, DCIS, with degree of loss correlating
with breast cancer progression and corresponding to a decrease in
patient survival. Mechanisms for decreased expression include LOH
at the TGFBR3 gene locus and potential transcriptional downregula-
tion of TβRIII by elevated TGF-β levels in the breast tumor microen-
vironment. Finally, we established a functional role for loss of TβRIII
expression, as restoring TβRIII expression dramatically inhibited
tumor invasiveness in vitro and tumor invasion, angiogenesis, and
metastasis in vivo. Mechanistically, TβRIII appeared to function by
undergoing ectodomain shedding, with sTβRIII antagonizing TGF-β
signaling and reducing invasiveness and angiogenesis in vivo. Taken
together, these results support loss of TβRIII expression as a frequent
and important step in breast cancer progression, directly promoting
breast cancer invasion and metastasis.
The dichotomous role of TGF-β signaling in breast cancer develop-
ment has been experimentally verified in several murine models. Spe-
cifically, blocking TGF-β signaling in a series of human breast-derived
cell lines representing different stages in breast cancer progression
rendered premalignant cells tumorigenic, and low-grade tumori-
genic cells more invasive, while making high-grade tumorigenic
cells less metastatic (39). In addition, introduction of constitutively
active TβRI delayed oncogenic Neu-induced breast tumor onset but
enhanced the frequency of lung metastasis in transgenic mice, where-
as dominant-negative TβRII enhanced Neu-induced tumor onset but
decreased subsequent lung metastasis (40). Furthermore, inducing
expression of active TGF-β1 after primary breast tumor formation
dramatically enhanced lung metastasis in a murine breast cancer
model without a detectable effect on primary tumor size (41). Taken
together, the results of these studies suggest that TGF-β suppresses
breast cancer progression in the early stages, but enhances tumor
progression and metastasis in the later stages. Different explanations
for this dichotomous function have been proposed, including TGF-β
exerting tumor-suppressing effects on epithelial-derived tumor cells
and tumor-promoting effects on stromal cells (increased angiogen-
esis and immunosuppression, altered tumor cell–extracellular matrix
interactions to enhance invasion and metastasis) (6). However,
TβRIII attenuates Smad2 phosphorylation in vitro and in
vivo. (A) TβRIII-overexpressing and control MDA-MB231
cells were treated with TGF-β1 under the indicated con-
ditions, and cell lysates were analyzed with a phospho-
Smad2 (p-Smad2) antibody. (B) Cells were transfected
with pE2.1 and pSVβ vector. Luciferase activity was deter-
mined after 24 hours of TGF-β1 treatment (100 pM) and is
expressed as the fold induction over no TGF-β treatment
after adjusting for β-galactosidase expression. This assay
was performed in triplicate at least 3 times. *P < 0.05. (C)
Phosphorylated Smad2 immunostaining of tissue sections
from 4T1-Neo and 4T1-TβRIII primary tumors. Represen-
tative results are shown. Note the significant decrease in
staining intensity in the 4T1-TβRIII tumor. Original magni-
Low levels of TβRIII predict decreased recurrence-free survival in
women with breast cancer. Five-year recurrence-free survival for
breast cancer with high or low TβRIII expression was analyzed based
on a microarray data set containing 286 patients. *P < 0.05.
?The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 117? ? ? Number 1? ? ? January 2007
emerging evidence suggests that TGF-β may exert its dichotomous
effects during carcinogenesis at least in part through biphasic effects
on the epithelial derived cancer cells themselves, as the cells alter their
molecular profiles to differentially respond to TGF-β (6). Thus, even
though resistant to the tumor suppressor effects of TGF-β during
tumorigenesis (growth inhibition, apoptosis, and differentiation),
the cancer cells may respond to TGF-β with increased motility and
invasiveness. Based on the present findings, we propose that loss of
TβRIII expression may be a mechanism for this differential response
to TGF-β during mammary carcinogenesis.
How might loss of TβRIII expression alter cellular responses to
TGF-β during mammary carcinogenesis? Although TβRIII was the
first TGF-β receptor cloned, as it has a short cytoplasmic domain
with no intrinsic kinase activity, its role in TGF-β signaling has not
been well characterized. TβRIII has classically been thought to act
as a TGF-β coreceptor, concentrating ligand on the cell surface and
enhancing ligand binding to the signaling TGF-β receptor TβRII
(42). However, emerging evidence supports a more substantial role
for TβRIII in regulating and mediating TGF-β signaling. TβRIII
has essential roles in chick (12) and murine development, with the
TβRIII knockout mouse having an embryonic lethal phenotype (13).
In addition, we have previously established that regulating TβRIII
expression is sufficient to alter TGF-β signaling (26), that the short
cytoplasmic domain of TβRIII is phosphorylated by TβRII (14) and
interacts with the PDZ domain–containing protein GIPC to sta-
bilize TβRIII expression on the cell surface and increasing TGF-β
signaling (26) as well as with the scaffolding protein β-arrestin2 to
mediate internalization of TβRIII and TβRII and downregulation
of TGF-β signaling (15). In addition, TβRIII undergoes ectodomain
shedding that releases the soluble extracellular domain (sTβRIII),
which has been demonstrated to effectively neutralize TGF-β and
antagonize autocrine TGF-β signaling. In breast cancer models,
expressing sTβRIII has been demonstrated to decrease tumorige-
nicity and spontaneous lung metastasis in immunocompromised
mice through effects on both the tumor cells (decreasing cell
growth and increasing apoptosis) (43) and the stroma (decreasing
angiogenesis) (28, 29). While our results in the immunocompetent
4T1 model confirm the effects of TβRIII on angiogenesis, we found
no significant effect of TβRIII expression on cellular proliferation
or apoptosis in either primary tumor or distant tumor metastastic
lesions in vivo. Instead, in addition to decreased angiogenesis, the
major effect of TβRIII in vitro and in vivo was to decrease cellular
invasiveness, with this effect mediated at least in part through the
production of sTβRIII. Therefore, we propose a model in which loss
of TβRIII expression results in alterations in TGF-β responsiveness
in both a cell-autonomous fashion (resulting in relative resistance
of breast cancer cells to TGF-β) and a non–cell-autonomous fash-
ion (by decreasing production of sTβRIII), effectively increasing
TGF-β signaling in both the cancer cells and the stromal elements.
Our in vitro and in vivo results demonstrating decreased Smad2
phosphorylation and decreased TGF-β responsiveness in the pres-
ence of TβRIII suggest that non–cell-autonomous regulation by
sTβRIII may have a dominant role in both tumor and stromal com-
partments. The contribution of TβRIII and sTβRIII on the balance
of TGF-β signaling and responsiveness in epithelial and stromal
compartments remains an area of active investigation.
TβRIII is located on chromosome 1p32, a region that frequently
exhibits LOH in a wide variety of human cancers, including breast,
colon, endometrial, gastric, kidney, lung, ovarian, and testicular
cancer (20–22). For breast cancer, LOH at 1p32 is associated with
a poorer prognosis (20, 21). Previous studies have examined several
potential tumor suppressor genes in this region, including mam-
mary-derived growth inhibitor (44) and TP73 (45); however, expres-
sion and functional studies did not provide sufficient evidence
supporting their role as tumor suppressor genes in breast cancer.
In the present study, LOH analysis revealed allelic imbalance at
the TβRIII loci in 50% of the patients, with LOH correlating with
loss of TβRIII expression. The observed decrease in TβRIII mRNA
and protein expression could result from haploid insufficiency,
as previously reported for TGF-β1 (46), or from transcriptional
downregulation or promoter hypermethylation of the remaining
allele. The current data strongly support TβRIII as a suppressor of
breast cancer progression. TβRIII has also been reported to be lost
at an early stage in renal cell carcinogenesis (47). Whether TβRIII
functions as a suppressor of cancer progression in renal cell and
other human cancers remains to be discerned.
Although breast cancer is thought to progress from a preinva-
sive state (DCIS) to invasive disease, we currently cannot determine
which DCIS lesions are likely to remain indolent, and thus may be
treated by local resection only, versus those DCIS lesions that will
progress to invasive disease and/or recur, necessitating more aggres-
sive treatment (i.e., postresection radiation, mastectomy, or adjuvant
hormonal or chemotherapy). Clearly, understanding the molecular
mechanisms by which DCIS becomes invasive and ultimately meta-
static will allow identification of patients at low or high risk of recur-
rence and invasion/metastasis and guide these treatment options. In
the present study, our data support loss of TβRIII expression in DCIS
as a common event potentially resulting in invasive and metastatic
disease. Thus, as would be predicted, later-stage invasive cancers have
a significantly higher frequency of TβRIII loss, and lower TβRIII
expression correlates with a poorer prognosis for patients with inva-
sive breast cancer. As this retrospective analysis was performed on
patient tumor samples that were heterogeneous for both tumor and
surrounding stromal tissue, we cannot be certain whether the loss in
TβRIII expression was in the tumor, stroma, or both, although our
own IHC analysis of a large tissue array support that loss was pri-
marily in tumor cells. Whether TβRIII-negative DCIS lesions have a
worse natural history and thus warrant more aggressive intervention
than TβRIII-positive DCIS lesions requires prospective validation.
TβRIII gene expression analysis on cDNA filter array. A filter array containing
normalized cDNA from 50 breast cancers and corresponding normal tis-
sues (Cancer Profiling Array; Clontech; Takara Bio Co.) was probed with
[32P]-labeled cDNA probes for TβRIII following methods recommended
by the manufacturer. In the 50 breast cancer samples, 33 were ductal
carcinoma, 10 were lobular carcinoma, and 2 were tubular carcinoma;
the remaining samples consisted of 1 each of mixed lobular/ductal car-
cinoma, medullary carcinoma, mucinous adenocarcinoma, fibrosarcoma,
and DCIS. The TβRIII cDNA probe was PCR amplified using the forward
primer GTAGTGGGTTGGCCAGATGGT and reverse primer CTGCT-
GTCTCCCCTGTGTG. Purified PCR products (25 ng) were labeled by
random primed DNA labeling using [α-32P]dCTP following the manufac-
turer’s protocol (Roche Diagnostics). Labeled cDNA probe was purified
on a BD CHROMA SPIN+STE-100 column (BD Biosciences — Clontech).
Images were acquired using a phosphorimager, and subsequent data analy-
sis was performed using NIH ImageJ software (http://rsb.info.nih.gov/ij/).
A normal/tumor ratio of 2 or higher was considered to be significant.
Breast cancer tissue array. A polyclonal antibody recognizing TβRIII pro-
tein was custom made by immunizing rabbits with a GST-fusion protein
216?The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 117? ? ? Number 1? ? ? January 2007
of the entire cytoplasmic domain of human TβRIII. The IgG fraction
of pre-immune and immune rabbit serum was collected using Immuno-
Pure IgG Purification kit (Pierce Biotechnology), and the specificity of
the antibody was established by comparing staining of the breast cancer
tissue arrays (Cooperative Breast Cancer Tissue Resource; National Can-
cer Institute) with preimmune serum and with the immune serum under
identical conditions, by the specific pattern of staining of cells known to
express TβRIII (breast epithelial cells) and lack of staining of cells known
not to express TβRIII (lymphocytes), and by Western blot using protein
extract from cell lines overexpressing human TβRIII. Breast cancer tissue
arrays (Cooperative Breast Cancer Tissue Resource) were deparaffinized,
rehydrated, treated with 3% hydrogen peroxide, and then blocked with
10% goat serum. The arrays were incubated with anti-TβRIII antibody
overnight at 4°C, washed in PBS, and further incubated with HRP-
conjugated anti-rabbit IgG (Vector Laboratory). Counterstaining was
performed using hematoxylin. As a negative control, duplicate sections
were immunostained with IgG purified from prebleed rabbit serum. The
immunoreactivity for TβRIII in breast epithelial and breast cancer cells
was relatively uniform within a specimen and was thus semiquantitative-
ly scored by staining intensity in a blinded manner with 0–1 defined as
no or weak staining, 2–3 as moderate staining, and 4–5 as intense stain-
ing. Standards for each staining score were used to maintain consistent
scoring across specimens.
LOH and sequence analysis. Genomic DNA extracted from human breast
cancer specimens and matching normal peripheral lymphocytes was kindly
provided by the Breast Cancer Tissue Repository at Duke University. Mic-
rosatellite markers D1S1588, D1S188, D1S2804, and D1S435 were used in
PCR reactions in which the forward primer was synthesized with a 5′ fluo-
rescent tag (Integrated DNA Technologies). PCR products were visualized
using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems), and data
were analyzed using GeneScan software (version 3.1; Applied Biosystems).
LOH was determined as at least a 50% reduction in the relative intensity of
one allele compared with the normal control. All samples positive for LOH
were independently analyzed twice. PCR products were also analyzed on 3%
MetaPhor agarose gels (Cambrex). For sequence analysis, 16 coding exons
of TβRIII were PCR amplified from the same DNA samples and subjected
to sequence analysis (Supplemental Table 1).
Real-time PCR. Cells were treated with 100 pM TGF-β1 for the times indi-
cated in Figures 3 and 8 and the Figure 7 legend. Total RNA was isolated
using RNeasy Mini Kit?(QIAGEN), and first-strand cDNA was synthesized
by M-MLV reverse transcriptase (Invitrogen). Quantitative real-time PCR
was performed in the presence of iQ SYBR Green Supermix (Bio-Rad) on a
Bio-Rad iCycler. Primer sequences are provided in Supplemental Table 2.
Relative levels were calculated using the comparative threshold cycle meth-
od, with data normalized to GAPDH and expressed relative to untreated
controls. All experiments were carried out in triplicate.
In vivo tumorigenicity and metastasis. Animal procedures were approved
by the Institutional Animal Care and Use Committee of Duke Univer-
sity. The 4T1-Luc cell line stably expressing firefly luciferase gene under
the selection of puromycin was generously provided by M.W. Dewhirst
(Duke University Medical Center). These cells were further transfected
with HA-tagged rat TβRIII under the selection of neomycin and expres-
sion confirmed by [125I]TGF-β1 binding and crosslinking. Cells were
implanted (50,000 cells/mouse) into the right-side axillary mammary
gland of 7-week-old virgin, female BALB/c mice (Charles River Laborato-
ries). Starting from day 10 after the implantation, growth of the tumors
was measured with a caliber in 2 dimensions on alternate days and
expressed as (length × width2) × 0.5. On day 20 after implantation, surgi-
cal resection of the primary tumors was performed under sterile condi-
tions. Four days after the surgery, tumor metastasis was recorded by bio-
luminescence imaging of the mice every 3 days for 19 days. Briefly, mice
were intraperitoneally injected with D-luciferin (Xenogen) at 150 μg/g.
Fifteen minutes following the injection, bioluminescence images were
acquired using a IVIS camera (Xenogen). Bioluminescence for ROI was
defined automatically, and data were expressed as photon flux (pho-
tons/s/cm2/steradian). Background photon flux was defined from a ROI
drawn over a mouse that was not given luciferin. At the end of the study
mice were sacrificed, and sites of metastasis were determined by visual
inspection. Interested organs were harvested for further IHC analysis and
RNA and protein extraction. We used 4T1-Luc cells, stably transfected
with the vector pcDNA3.1-Neo, in parallel as controls.
TUNEL, PCNA, CD31, and phosphorylated Smad2 immunostaining. TUNEL
and PCNA immunostainings were performed on paraffin-embedded tis-
sue sections according to the manufacturer’s instructions (TUNEL, In Situ
Cell Death Detection kit, POD; Roche Diagnostics; PCNA, Santa Cruz Bio-
technology Inc.). CD31 immunostaining was performed on frozen tissue
sections as specified by the manufacturer (Cell Signaling Technology). For
phosphorylated Smad2 staining, antigen retrieval was carried out by boil-
ing slides in 10 mM citrate buffer (pH 6.0) for 5 minutes in a microwave
after blocking in 3% hydrogen peroxide.
Matrigel invasion assay. We seeded 75,000 cells in the Matrigel-coated
upper chamber (BD Biosciences) of a 24-well transwell. Media containing
10% FBS was placed in the lower chamber as a chemoattractant. After 2
hours’ incubation, 15 pM TGF-β1 was added into the designated upper
chambers. Twenty-four hours later, the cells on the upper surface of the
filter were removed by gently scrubbing with a cotton swab. The cells
that migrated to the underside of the filter were fixed and stained with
H&E. Each filter was removed and examined microscopically, and 3 ran-
dom images were acquired. Cells present in each image were counted. In
some assays, conditioned serum-free medium collected from COS-7 cells
transiently transfected with empty vector, full-length TβRIII, or sTβRIII
construct was used to resuspend cells to be seeded in the upper chambers.
These assays were performed in triplicate at least 3 times.
TGF-β binding and crosslinking assay. We incubated 100 pM [125I]TGF-β1 with
500 μl of the cell medium in the presence of protease inhibitors for 3 hours at
4°C. The [125I]TGF-β1–sTβRIII complex was then crosslinked with 0.5 mg/ml
disuccinimidyl suberate and immunoprecipitated with a polyclonal anti-
body recognizing the extracellular domain of TβRIII (R&D Systems). The
final complex was visualized after SDS-PAGE and autoradiography.
Transcription reporter luciferase assays. Cells were transfected with a
pE2.1 vector that contains the luciferase gene under the regulation of a
promoter based on the TGF-β–inducible promoter PAI-1 and the pSVβ
vector encoding β-galactosidase as a control for transfection efficien-
cy. After 24 hours, the cells were treated with TGF-β1 (100 pM) for an
additional 24-hour period. The cells were lysed in luciferase lysis buffer
(Promega). The luciferase activity was read after the addition of luciferin
(Promega) using an automated luminometer. The luciferase activity was
expressed as the fold induction over no TGF-β treatment after adjusting
for β-galactosidase expression.
[3H]Thymidine incorporation assay. Cells growing in 24-well plates were
treated with 0–200 pm TGF-β1 for 48 hours when they reached 80% con-
fluency and then incubated with 10 μCi of [3H]thymidine (Amersham Bio-
sciences) for an additional 4 hours. Cells were washed with PBS and 5%
trichloroacetic acid before being harvested with 0.1 N NaOH. The amount
of incorporated [3H]thymidine was determined by scintillation counting.
Growth inhibition was calculated as the ratio of radioactivity with TGF-β1
treatment to radioactivity without TGF-β1 treatment.
Statistics. Statistical analysis was performed using the 2-tailed Student’s t
test unless otherwise indicated. All data are presented as mean ± SEM. P val-
ues less than 0.05 were considered to be significant.
?The?Journal?of?Clinical?Investigation? ? ? http://www.jci.org? ? ? Volume 117? ? ? Number 1? ? ? January 2007
Tissue microarray slides were provided by the Cooperative Breast Can-
cer Tissue Resource, which is funded by the National Cancer Institute.
We thank M.W. Dewhirst for the 4T1-Luc cell line. We thank N. Glov-
er and J. Fuller for technical assistance. These studies were supported
by National Institutes of Health/National Cancer Institute grant
R01-CA106307 (to G.C. Blobe), by a postdoctoral fellowship from
the Susan G. Komen Breast Cancer Foundation (to M. Dong), and
by predoctoral fellowships from the Department of Defense Breast
Cancer Research Program (to K.C. Kirkbride and J.D. Lee).
Received for publication June 5, 2006, and accepted in revised form
October 10, 2006.
Address correspondence to: Gerard C. Blobe, 221B MSRB
Research Drive, Box 2631 DUMC, Durham, North Carolina
27710, USA. Phone: (919) 668-1352; Fax: (919) 668-2458; E-mail:
Tam How, Kellye C. Kirkbride, Kelly J. Gordon, Jason D. Lee, and
Nadine Hempel contributed equally to this work.
1. Massague, J. 1998. TGF-beta signal transduction.
Annu. Rev. Biochem. 67:753–791.
2. Jhappan, C., et al. 1993. Targeting expression of a
transforming growth factor beta 1 transgene to the
pregnant mammary gland inhibits alveolar devel-
opment and lactation. EMBO J. 12:1835–1845.
3. Pierce, D.F., Jr., et al. 1993. Inhibition of mammary
duct development but not alveolar outgrowth dur-
ing pregnancy in transgenic mice expressing active
TGF-beta 1. Genes Dev. 7:2308–2317.
4. Pierce, D.F., Jr., et al. 1995. Mammary tumor sup-
pression by transforming growth factor beta 1
transgene expression. Proc. Natl. Acad. Sci. U. S. A.
5. Decensi, A., et al. 1998. Correlation between plasma
transforming growth factor-beta 1 and second pri-
mary breast cancer in a chemoprevention trial. Eur.
J. Cancer. 34:999–1003.
6. Elliott, R.L., and Blobe, G.C. 2005. Role of trans-
forming growth factor Beta in human cancer.
J. Clin. Oncol. 23:2078–2093.
7. Walker, R.A., and Dearing, S.J. 1992. Transforming
growth factor beta 1 in ductal carcinoma in situ
and invasive carcinomas of the breast. Eur. J. Cancer.
8. Dalal, B.I., Keown, P.A., and Greenberg, A.H. 1993.
Immunocytochemical localization of secreted
transforming growth factor-beta 1 to the advanc-
ing edges of primary tumors and to lymph node
metastases of human mammary carcinoma. Am. J.
9. Gorsch, S.M., Memoli, V.A., Stukel, T.A., Gold,
L.I., and Arrick, B.A. 1992. Immunohistochemical
staining for transforming growth factor beta 1
associates with disease progression in human
breast cancer. Cancer Res. 52:6949–6952.
10. Ghellal, A., et al. 2000. Prognostic significance of TGF
beta 1 and TGF beta 3 in human breast carcinoma.
Anticancer Res. 20:4413–4418.
11. Xie, W., et al. 2002. Alterations of Smad signaling
in human breast carcinoma are associated with
poor outcome: a tissue microarray study. Cancer
12. Brown, C.B., Boyer, A.S., Runyan, R.B., and Barnett,
J.V. 1999. Requirement of type III TGF-beta recep-
tor for endocardial cell transformation in the heart.
13. Stenvers, K.L., et al. 2003. Heart and liver defects and
reduced transforming growth factor beta2 sensitiv-
ity in transforming growth factor beta type III recep-
tor-deficient embryos. Mol. Cell. Biol. 23:4371–4385.
14. Blobe, G.C., et al. 2001. Functional roles for the
cytoplasmic domain of the type III transforming
growth factor beta receptor in regulating trans-
forming growth factor beta signaling. J. Biol. Chem.
15. Chen, W., et al. 2003. Beta-arrestin 2 mediates endo-
cytosis of type III TGF-beta receptor and down-reg-
ulation of its signaling. Science. 301:1394–1397.
16. Chen, C., Wang, X.F., and Sun, L. 1997. Expression
of transforming growth factor beta (TGFbeta) type
III receptor restores autocrine TGFbeta1 activity
in human breast cancer MCF-7 cells. J. Biol. Chem.
17. Sun, L., and Chen, C. 1997. Expression of transform-
ing growth factor beta type III receptor suppresses
tumorigenicity of human breast cancer MDA-MB-
231 cells. J. Biol. Chem. 272:25367–25372.
18. Parsons, R., et al. 1995. Microsatellite instability
and mutations of the transforming growth fac-
tor beta type II receptor gene in colorectal cancer.
Cancer Res. 55:5548–5550.
19. Hahn, S.A., et al. 1996. DPC4, a candidate tumor
suppressor gene at human chromosome 18q21.1.
20. Ragnarsson, G., et al. 1999. Loss of heterozygos-
ity at chromosome 1p in different solid human
tumours: association with survival. Br. J. Cancer.
21. Borg, A., Zhang, Q.X., Olsson, H., and Wenngren, E.
1992. Chromosome 1 alterations in breast cancer:
allelic loss on 1p and 1q is related to lymphogenic
metastases and poor prognosis. Genes Chromosomes
22. Bieche, I., Khodja, A., and Lidereau, R. 1999. Dele-
tion mapping of chromosomal region 1p32-pter in
primary breast cancer. Genes Chromosomes Cancer.
23. Ji, C., Chen, Y., McCarthy, T.L., and Centrella, M.
1999. Cloning the promoter for transforming
growth factor-beta type III receptor. Basal and con-
ditional expression in fetal rat osteoblasts. J. Biol.
24. Pulaski, B.A., and Ostrand-Rosenberg, S. 1998.
Reduction of established spontaneous mammary
carcinoma metastases following immunotherapy
with major histocompatibility complex class II
and B7.1 cell-based tumor vaccines. Cancer Res.
25. Heppner, G.H., Miller, F.R., and Shekhar, P.M.
2000. Nontransgenic models of breast cancer.
Breast Cancer Res. 2:331–334.
26. Blobe, G.C., Liu, X., Fang, S.J., How, T., and
Lodish, H.F. 2001. A novel mechanism for regulat-
ing transforming growth factor beta (TGF-beta)
signaling. Functional modulation of type III
TGF-beta receptor expression through interaction
with the PDZ domain protein, GIPC. J. Biol. Chem.
27. Lopez-Casillas, F., Payne, H.M., Andres, J.L., and
Massague, J. 1994. Betaglycan can act as a dual
modulator of TGF-beta access to signaling recep-
tors: mapping of ligand binding and GAG attach-
ment sites. J. Cell Biol. 124:557–568.
28. Bandyopadhyay, A., et al. 2002. Extracellular
domain of TGFbeta type III receptor inhibits angio-
genesis and tumor growth in human cancer cells.
29. Bandyopadhyay, A., et al. 2002. Antitumor activ-
ity of a recombinant soluble betaglycan in human
breast cancer xenograft. Cancer Res. 62:4690–4695.
30. Andres, J.L., Stanley, K., Cheifetz, S., and Massague,
J. 1989. Membrane-anchored and soluble forms
of betaglycan, a polymorphic proteoglycan that
binds transforming growth factor-beta. J. Cell Biol.
31. Philip, A., Hannah, R., and O’Connor-McCourt,
M. 1999. Ectodomain cleavage and shedding of the
type III transforming growth factor-beta receptor in
lung membranes effect of temperature, ligand bind-
ing and membrane solubilization. Eur. J. Biochem.
32. Cheung, H.K., Mei, J., and Xu, R.J. 2003. Quantifi-
cation of soluble betaglycan in porcine milk. Asia
Pac. J. Clin. Nutr. 12(Suppl.):S61.
33. van’t Veer, L.J., et al. 2002. Gene expression pro-
filing predicts clinical outcome of breast cancer.
34. Sorlie, T., et al. 2003. Repeated observation of
breast tumor subtypes in independent gene
expression data sets. Proc. Natl. Acad. Sci. U. S. A.
35. Ma, X.J., et al. 2004. A two-gene expression ratio
predicts clinical outcome in breast cancer patients
treated with tamoxifen. Cancer Cell. 5:607–616.
36. Wang, Y., et al. 2005. Gene-expression profiles to
predict distant metastasis of lymph-node-negative
primary breast cancer. Lancet. 365:671–679.
37. Moody, S.E., et al. 2005. The transcriptional repres-
sor Snail promotes mammary tumor recurrence.
Cancer Cell. 8:197–209.
38. Kamangar, F., Dores, G.M., and Anderson, W.F.
2006. Patterns of cancer incidence, mortality, and
prevalence across five continents: defining priorities
to reduce cancer disparities in different geographic
regions of the world. J. Clin. Oncol. 24:2137–2150.
39. Tang, B., et al. 2003. TGF-β switches from tumor sup-
pressor to prometastatic factor in a model of breast
cancer progression. J. Clin. Invest. 112:1116–1124.
40. Siegel, P.M., Shu, W., Cardiff, R.D., Muller, W.J., and
Massague, J. 2003. Transforming growth factor beta
signaling impairs Neu-induced mammary tumori-
genesis while promoting pulmonary metastasis.
Proc. Natl. Acad. Sci. U. S. A. 100:8430–8435.
41. Muraoka-Cook, R.S., et al. 2004. Conditional over-
expression of active transforming growth factor
beta1 in vivo accelerates metastases of transgenic
mammary tumors. Cancer Res. 64:9002–9011.
42. Lopez-Casillas, F., Wrana, J.L., and Massague, J.
1993. Betaglycan presents ligand to the TGF beta
signaling receptor. Cell. 73:1435–1444.
43. Lei, X., Bandyopadhyay, A., Le, T., and Sun, L. 2002.
Autocrine TGFbeta supports growth and survival of
human breast cancer MDA-MB-231 cells. Oncogene.
44. Huynh, H., Alpert, L., and Pollak, M. 1996. Silenc-
ing of the mammary-derived growth inhibitor
(MDGI) gene in breast neoplasms is associated with
epigenetic changes. Cancer Res. 56:4865–4870.
45. Ahomadegbe, J.C., et al. 2000. Loss of heterozygosity,
allele silencing and decreased expression of p73 gene
in breast cancers: prevalence of alterations in inflam-
matory breast cancers. Oncogene. 19:5413–5418.
46. Tang, B., et al. 1998. Transforming growth factor-
beta1 is a new form of tumor suppressor with true
haploid insufficiency. Nat. Med. 4:802–807.
47. Copland, J.A., et al. 2003. Genomic profiling iden-
tifies alterations in TGFbeta signaling through
loss of TGFbeta receptor expression in human
renal cell carcinogenesis and progression. Oncogene.