(Received for publication, October 28, 1996, and in revised form, December 30, 1996)
From the Departments of We have studied the role of autocrine
transforming growth factor- Transforming growth factor- Both signaling receptors seem to be needed for TGF- All three TGF- Normal and tumorigenic human breast epithelial cells in culture express
TGF- MCF-7 cells were provided by C. K. Osborne (University of Texas Health Science Center, San Antonio, TX)
and have been described previously (33). T47D cells were from ATCC
(Rockville, MD). They were both cultured in IMEM (Life Technologies,
Inc.) supplemented with 5% (MCF-7) or 10% (T47D) fetal calf serum
(JRH Biosciences, Lenexa, KS) and 10 nM insulin. The late
passage MCF-7 cells transfected with a TGF- Secreted TGF- Binding was performed on intact adherent cells in
100-mm tissue culture dishes. Cells were incubated in binding buffer
(see above) containing 1 ng/ml 125I-TGF- Cells were trypsinized,
suspended in cold PBS, and fixed in absolute ethanol (final
concentration 67%). After overnight refrigeration, DNA was stained in
the dark with 50 µg/ml of propidium iodide (PI) containing 125 units/ml protease-free RNase (Calbiochem), both in PBS. DNA histograms
were analyzed in a FACScan flow cytometer (Beckton Dickinson,
Mansfield, MA) and modeled off-line using Modfit software (Verity,
Topsham, ME).
Cells (2 × 104/well) were plated in 24-well dishes in regular growth
medium. The following day, 1 µM tamoxifen or TGF- Adherent monolayers were treated with tamoxifen or TGF- We first examined the modulation
of TGF-
Tamoxifen increases the secretion of TGF- Subconfluent exponentially growing breast cancer cells in 100-mm tissue
culture dishes were washed with serum-free IMEM and incubated overnight
in serum-free IMEM containing 0.1% ethanol or 1 µM
tamoxifen. The cell CM was collected after 24 h, acidified with 1 N HC1 to pH 1.5 for 1 h on ice, and reneutralized to
pH 7.6 with 1 N NaOH. TGF- Medicine and
Cell Biology,
(TGF-
) signaling on
antiestrogen-mediated growth inhibition of
hormone-dependent T47D and MCF-7 human breast carcinoma
cells. Tamoxifen treatment increased the secretion of TGF-
activity into serum-free cell medium and the cellular content of affinity cross-linked type I and III TGF-
receptors in both cell lines. Anti-pan-TGF-
antibodies did not block anti-estrogen-induced recruitment in G1 and inhibition of
anchorage-dependent and -independent growth of both cell
lines. Early passage MCF-7 cells, which exhibit detectable type II
TGF-
receptors at the cell surface and exquisite sensitivity to
exogenous TGF-
1, were transfected with a tetracycline-controllable dominant-negative TGF-
RII (
RII) construct. Although the TGF-
1 response was blocked by removal of tetracycline in MCF-7/
RII cells,
tamoxifen-mediated suppression of Rb phosphorylation, recruitment in
G1, and inhibition of cell proliferation were identical in the presence and absence of tetracycline. TGF-
1 treatment
up-regulated the Cdk inhibitor p21 and induced its association with
Cdk2 in MCF-7 cells; these responses were blocked by the
RII
transgene product. In MCF-7 cells with a functional TGF-
signaling
pathway, tamoxifen did not up-regulate p21 nor did it induce
association of p21 with Cdk2, suggesting alternative mechanisms for
antiestrogen-mediated cytostasis. Finally, transfection of
late-passage, TGF-
1 unresponsive MCF-7 cells with high levels of
TGF-
RII restored TGF-
1-induced growth inhibition but did not
enhance tamoxifen response in culture. Taken together these data
strongly argue against any role for TGF-
signaling on
tamoxifen-mediated growth inhibition of hormone-dependent breast cancer cells.
s
(TGF-
s)1 are potent regulators of
cellular proliferation, differentiation, morphogenesis as well as
extracellular matrix formation, extracellular proteolysis, and
inflammation (1-3). In epithelial cells a major effect of TGF-
is
its ability to inhibit cell proliferation (4). Three different
mammalian TGF-
isoforms (TGF-
1, -
2, and -
3) encoded by
different genes have been identified, and they exhibit similar effects
in a variety of biological assays (5). Three membrane ligand-binding
proteins with sizes of 53, 73, and ~250 kDa have been reported as
TGF-
receptors type I, II, and III, respectively. Type I and II
receptors are transmembrane serine/threonine kinases directly involved
in signal transduction, while the type III receptor functions mainly by
presenting the ligand to the signaling type I and II receptors and as a
storage protein (6, 7). Both TGF-
and its receptor molecules are
expressed ubiquitously by normal and transformed cells.
responsiveness
(8), and type II receptor expression correlates with the
anti-proliferative activity of TGF-
(9, 10). TGF-
arrests cell
growth in the G1 phase of the cell cycle and most probably affects multiple signaling pathways (11). TGF-
has been shown to
retain the retinoblastoma susceptibility gene (pRb) in a
hypophosphorylated form, which prevents cells from entering the S phase
(12). By regulating the formation of Cdk-cyclin complexes and their
inhibitor levels, TGF-
contributes to the accumulation of
hypophosphorylated pRb (13). In several cell lines, TGF-
1 also
induces rapid down-regulation of c-myc expression (14, 15),
suggesting this is an additional mechanism by which these peptides
suppress cell growth.
isoforms are expressed in mouse mammary gland, and
there are data supporting their role in the development of the mouse
mammary gland (16, 17). Exogenous TGF-
administrated by slow release
pellets or tissue-specific expression of active TGF-
1 in the mammary
gland of transgenic mice leads to ductal hypoplasia and suppression of
ductal branching (16, 18, 19).
1 mRNA and secrete TGF-
receptor binding activity into
their medium (20, 21). Published data support the notion that
endogenous TGF-
s function as autocrine growth regulators of breast
cancer cell proliferation (22, 23). Antibodies that neutralize mature
TGF-
s stimulate the proliferation of estrogen-independent breast
cancer cell lines (23). Growth stimulation of
estrogen-dependent breast cancer cells with estradiol or
the testosterone derivative norethindrone is associated with
down-regulation of TGF-
2 and -
3 mRNAs (24-26). Growth
inhibition of these cell lines by the antiestrogens tamoxifen or
toremifene and the progestin analogue gestodene is associated with
enhanced TGF-
1 mRNA expression or increased secretion of TGF-
bioactivity or protein synthesis without associated mRNA changes
(22, 27, 28), thus leading to the hypothesis that autocrine TGF-
signaling contributed to antiestrogen's actions. Some reports,
however, argue against TGF-
s' role in the growth inhibitory
response to antiestrogens. MCF-7 and T47D breast cancer cells can
exhibit resistance to TGF-
1-mediated growth inhibition despite
retaining sensitivity to tamoxifen (29, 30). In addition, T47D and
CAMA-1 breast cancer lines, which lack mRNA for TGF-
RII and
hence response to exogenous TGF-
1, remain sensitive to the
cytostatic effect of tamoxifen (31, 32). By using anti-TGF-
neutralizing antibodies as well as dominant negative TGF-
RII
constructs in estrogen-dependent, tamoxifen-sensitive human
breast cancer cells, we have formally tested in this study the role of
endogenous TGF-
signaling on the cellular response to antiestrogens
in breast carcinoma.
Cell Lines and Antibodies
RII expression vector
(MCF-7/RII) were described previously (9). These cells as well as early
passage MCF-7 cells transfected with a dominant negative
tetracycline-repressible type II TGF-
receptor
(MCF-7/
RII)2 were cultured in IMEM
containing 10% FCS and 500 µg/ml G418 (Life Technologies, Inc.). The
-human pRb monoclonal antibody was from PharMingen (San Diego, CA).
The 2G7, 12H5, and 4A11 antibodies (provided by B. M. Fendly,
Genentech, South San Francisco, CA) were raised against human
recombinant TGF-
1 and have been characterized previously (34). The
12H5 IgG2 is devoid of TGF-
neutralizing activity, while
the 4A11 IgG1 and the 2G7 IgG2 neutralize the growth inhibitory activity of TGF-
1 and TGF-
1, -
2, and -
3 on Mv1Lu mink lung epithelial cells, respectively (34). The Cdk2
polyclonal IgG (M2), raised against carboxyl-terminal residues 283-298, and the p27 polyclonal IgG (C-19) were from Santa Cruz Biotechnology (Santa Cruz, CA). The p21WAF1/CIP1 monoclonal
antibody was purchased from Oncogene Science (Cambridge, MA). The
polyclonal antibody C-16 (Santa Cruz Biotechnology) was used for
immunoprecipitation of TGF-
RII.
Radioreceptor Assay
bioactivity was measured in
serum-free IMEM conditioned for 24 h by adherent breast cancer
cells as described previously (23). When indicated, the CM was
acidified with 1 N HCl to pH 1.5 for 1 h at 4 °C
and reneutralized with 1 N NaOH before testing. CM was then
tested in a TGF-
radioreceptor assay (23) utilizing AKR-2B mouse
fibroblasts as indicator cells. Binding was performed in six-well
plates with 1 ml/well binding buffer (128 mM NaCl, 5 mM KCl, 5 mM MgSO4, 1.2 mM CaCl2, 50 mM Hepes, pH 7.5, 0.2% BSA) containing 0.25 ng/ml 125I-TGF-
1 (specific
activity 173 µCi/µg; DuPont NEN) with or without variable volumes
of CM for 4 h at 4 °C. Human recombinant TGF-
1 (Genentech)
was used to generate a standard curve from which the receptor binding
activity in CM was calculated. In this assay, TGF-
1 and TGF-
2 are
equipotent in displacing 125I-TGF-
1 binding (35).
Addition of 1 µM tamoxifen directly to the radioreceptor
assay has no effect on TGF-
1 binding by AKR-2B cells.3
Binding and Affinity
Cross-linking
1 with or
without 100-fold excess unlabeled TGF-
1 for 4 h at 4 °C with
gentle rocking. After two washes with ice-cold binding buffer without
BSA on ice, the bound 125I-TGF-
1 was cross-linked to
cell surface receptors with 50 µM disuccinimidyl suberate
(Pierce) for 15 min at 4 °C in 10 ml of binding buffer without BSA.
Cells were then scraped and solubilized as described previously (23).
The samples were subjected to 5-10% gradient SDS-PAGE and labeled
receptors visualized by autoradiography. In some cases, to enhance the
sensitivity of the assay, approximately 5 × 107 cells
were labeled in 1 ml of binding buffer with 5 ng/ml (0.2 nM) 125I-TGF-
1 and cross-linked under the
same conditions as above. After solubilization, cross-linked cell
lysates were precipitated overnight at 4 °C with the C-16 TGF-
RII
polyclonal antibody (Santa Cruz Biotechnology) followed by protein
A-Sepharose for 1 h. Immune complexes were then resolved by
5-10% gradient SDS-PAGE and visualized by autoradiography.
1 was added. After 4-5 days, cells from triplicate samples were assessed in
a Coulter Z1 counter (Coulter Electronics Limited, Beds., United Kingdom). For testing of anchorage-independent growth, a 1-ml top layer
containing a single-cell suspension of 3 × 104 cells,
0.8% agarose (Sea-Plaque, FMC Corp. BioProducts, Rockland, ME), IMEM,
10% FCS, and 10 mM Hepes, with or without different concentrations of TGF-
1 or 1 µM tamoxifen was added to
a 1-ml bottom layer of 0.8% agarose, 10% FCS in 35-mm dishes. Dishes were incubated in a humidified 5% CO2 incubator at
37 °C, and colonies measuring
50 µm counted after 10 days using
an Omnicon Stem Model II Image Analyzer (Bausch & Lomb, Rochester,
NY).
1
for 18 h. Cells were then washed twice with ice-cold PBS and lysed with EBC buffer (50 mM Tris-HCl, pH 8.0, 120 mM
NaCl, 0.5% Nonidet P-40, 100 mM NaF, 200 µM
Na3VO4, and 10 µg/ml each aprotinin, leupeptin, and phenylmethanesulfonyl fluoride) for 30 min at 4 °C. The lysates were collected and cleared from detergent-insoluble material by 10,000 × g centrifugation. Equal aliquots
of protein (BCA method, Pierce) were separated by 8% (pRb) or 12%
(p21 and p27) SDS-PAGE, transferred to nitrocellulose membranes by
semidry electrophoretic transfer (Bio-Rad), and subjected to immunoblot analysis with an
-human pRb, p21, or p27 monoclonal antibodies. Nonspecific binding was blocked with 5% nonfat milk in
Tween/Tris-buffered saline (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature.
Bound antibodies were detected using horseradish peroxidase-conjugated
anti-mouse Ig (Amersham) and enhanced chemiluminescence (KPL,
Gaithersburg, MD). In some cases, 300 µg of protein were precipitated
overnight with 2 µg of a Cdk2 polyclonal antibody at 4 °C followed
by protein A-Sepharose (Sigma) for 1 h. The
Sepharose beads were washed three times with RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet
P-40, 0.5% sodium deoxycholate, 0.1% SDS). Immune complexes were next solubilized in Laemmli sample buffer, boiled, resolved by SDS-PAGE, and
subjected to a p21 immunoblot procedure.
Tamoxifen Increases Secreted TGF- Bioactivity and TGF-
Binding in MCF-7 and T47D Cells
secretion by tamoxifen in the ER-positive MCF-7 and T47D
human breast carcinoma cell lines. Exponentially growing cells were
treated with 0.1% ethanol (controls) or 1 µM tamoxifen
for 24 h in serum-free medium. TGF-
activity was measured in a
125I-TGF-
1 radioreceptor assay. In the absence of acid
activation in vitro, TGF-
activity was below the
detection limit of the binding assay, indicating that the majority of
the secreted TGF-
was in a latent form. Tamoxifen induced increases
of approximately 30- and 4-fold in the secretion of acid-activable
TGF-
activity in MCF-7 and T47D cultures, respectively (Table
I).
activity by MCF-7 and
T47D cells
activity in CM was measured
in a 125I-TGF-
1 radioreceptor assay with AKR-2B mouse
fibroblasts as indicator cells. TGF-
1 equivalents of receptor
binding activity in CM were extrapolated from a competition standard
curve with recombinant unlabeled TGF-
1 and standardized to cell
number.
Treatment
TGF-
a
MCF-7
T47D
Control (0.1% EtOH)
0.06
0.22
Tamoxifen (1 µM)
1.85
0.87
a
The results are expressed as nanograms of TGF- 1
equivalents/106 cells/24 h.
To test whether endogenous TGF- ligands in response to the
antiestrogen were masking endogenous TGF-
receptors, we examined the
effect of tamoxifen on 125I-TGF-
1 binding and the
cellular content of affinity cross-linked TGF-
receptors. Both MCF-7
and T47D cells exhibit type I and type III TGF-
receptors at the
cell surface, whereas TGF-
RII was undetectable (Fig.
1). To enhance the sensitivity of the assay, we labeled
5 × 107 cells with 10 ng/ml 125I-TGF-
1
in a 1-ml suspension followed by cross-linking and immunoprecipitation with TGF-
RII antibodies, but were still unable to detect any binding
by type II receptors at 4 °C (data not shown). Treatment of cells
with 1 µM tamoxifen for 3 days up-regulated type I and III TGF-
receptors in both cell lines (Fig. 1). This up-regulation was first obvious at 48 h and was maximal at 72-96 h. Bound cpm corrected for protein content in cross-linked cell lysates were
5-fold higher in tamoxifen-treated than in control lysates. A mild
acid wash (pH 2.8) modestly increased 125I-TGF-
1 binding
in control and tamoxifen-treated cells,3 arguing against
significant masking of TGF-
binding sites by autocrine ligands in
the presence or absence of the antiestrogen.
Neutralizing Anti-TGF-
To determine whether the enhanced TGF-
secreted activity was mediating tamoxifen actions, we performed studies
with anti-TGF-
antibodies. These antibodies have been shown to
stimulate the proliferation of breast tumor cells with an operative
autocrine TGF-
pathway by neutralizing endogenous mature TGF-
s
(23). Cells were treated with 1 µM tamoxifen or 0.1%
ethanol (control) in the presence of IgGs (100 µg/ml) for 3 days and
cell cycle distribution analyzed by flow cytometry of PI-labeled DNA.
In both lines, tamoxifen induced a marked decrease of the percentage of
cells in S phase and an accumulation in G1. Neither the
anti-pan-TGF-
2G7 or the anti-TGF-
1 4A11 antibodies nor their
respective controls, the 12H5 IgG2 and an irrelevant
IgG1, altered tamoxifen-mediated cytostasis (Fig.
2A). Identical results were obtained in a
colony-forming soft agarose assay. In this assay, both MCF-7 and T47D
cells were markedly inhibited by the antiestrogen; this inhibition was
not altered by any of the TGF-
antibodies (Fig. 2B). By
themselves, the IgGs utilized had no growth effects on either cell line
(data not shown).
High Expression of Functional TGF-
Transfection with high levels of a
tetracycline-repressible TGF-RII expression vector into MCF-7 cells
(MCF-7/RII cells, Ref. 9) restores sensitivity to growth inhibition by
exogenous TGF-
. Therefore, in these cells, we tested whether
up-regulation of an operative TGF-
signaling pathway would enhance
antiestrogen-induced growth inhibition. MCF-7/RII cells were plated
with or without 0.1 µM tetracycline. The following day, 1 µM tamoxifen or 0.1% EtOH were added to the monolayers
and cell proliferation assessed 4 days later. There was a similar 60%
inhibition of growth in tamoxifen-treated monolayers relative to
controls in the absence or presence of transfected TGF-
RII. Similar
to its effect on endogenous TGF-
RI and TGF-
RIII in MCF-7 and T47D
cells (Fig. 1), tamoxifen also increased the cellular content of
TGF-
RII in MCF-7/RII cells with a simultaneous up-regulation of type
I receptor sites (Fig. 3B). Similar to the
parental MCF-7 cells (Table I), incubation of MCF-7/RII cells with 1 µM tamoxifen increased the secretion of TGF-
activity
from 0.1 to 1.5 ng/106 cells/24 h as measured by
radioreceptor assay of conditioned medium. Since these cells have an
impaired ability for anchorage-independent growth, soft agarose
colony-forming assays to test the modulation of tamoxifen sensitivity
by TGF-
RII were not useful.
Expression of a Dominant Negative TGF-
Since the
neutralizing TGF- antibodies may not be effective in blocking a
potential ligand/receptor intracellular coupling or a direct
ligand-independent effect of tamoxifen on TGF-
RII, we used a
dominant negative approach to block TGF-
RII function in a
cell-autonomous experimental system. For this purpose we used early
passage MCF-7 breast cancer cells, which, different than the late
passage cells used above, exhibit detectable TGF-
RII protein at the
cell surface and are markedly growth inhibited by exogenous TGF-
s
(36). Expression of a tetracycline-repressible dominant negative type
II TGF-
receptor in these cells (MCF-7/
RII) blocks cellular
responses to exogenous TGF-
1.2 We first studied the
proliferation effects of prolonged tamoxifen treatment in cells
preincubated (for 24 h) or not with 0.1 µM tetracycline, concentration known to maximally induce the
RII mutant
protein.2 Both anchorage-dependent and
-independent proliferation were inhibited by 1 µM
tamoxifen in the presence or absence of tetracycline (Fig.
4). Similar results were obtained with 5-10
µM amounts of the antiestrogen toremifene (27) in
monolayer culture (data not shown).
Both TGF-1 (4, 12) and antiestrogens (38) can recruit sensitive
cells in the G1 phase of the cell cycle while suppressing Rb phosphorylation (12, 39). Therefore, we studied the impact of the
RII mutant on tamoxifen-mediated cell cycle arrest and Rb
phosphorylation in MCF-7/
RII cells. A 72-h incubation with 1 µM tamoxifen markedly increased the proportion of cells
in G1, while reducing those in G2M and S phases
of the cycle. These changes in response to antiestrogen were almost
identical in the presence or absence of tetracycline (Fig.
5). Consistent with this result, a 24-h incubation with
1 µM tamoxifen reduced Rb hyperphosphorylation in
MCF-7/
RII cells under conditions in which the
RII mutant type II
receptor was expressed or not (Fig. 6) further arguing against any role for endogenous TGF-
signaling on
antiestrogen-mediated G1 arrest.
TGF-
TGF-1 has been shown to inhibit the kinase
activity of cyclin E-Cdk2 complexes (7) and hence suppress Rb
phosphorylation. One reported mechanism for such inhibition is
induction of the Cdk inhibitor p21, which then associates with the
cyclin E-Cdk2 complex and inhibits its kinase (40, 41). A similar
induction of p21 has been reported recently by the anti-estrogen
ICI182780 in MCF-7 breast cancer cells (39). Therefore, we examined
whether TGF-
1 and tamoxifen suppressed Rb phosphorylation by
inducing p21 in MCF-7/
RII cells. An overnight incubation with 1 ng/ml TGF-
1 in the presence of tetracycline markedly up-regulated
p21 protein levels and induced its association with Cdk2, as supported by coprecipitation of p21 with Cdk2 antibodies. This induction and
association with Cdk2 were abrogated by removal of tetracycline, which
eliminates endogenous TGF-
RII signaling (Fig. 7).
Interestingly, 1 µM tamoxifen did not induce p21 nor did
it induce its association with Cdk2, suggesting its suppressive action
on Rb phosphorylation is mediated by a mechanism(s) other than
up-regulation of autocrine growth inhibitory TGF-
s. Prolongation of
tamoxifen treatment to 72 h still did not induce p21 protein. A
24-h treatment of MCF-7/
RII cells with 1 µM tamoxifen
or 1 ng/ml TGF-
1 in the presence or absence of tetracycline, did not
induce the Cdk inhibitor p27 as measured by immunoblot analysis (data
not shown).
It is proposed that antiestrogens induce growth inhibition of
human breast tumor cells by up-regulating expression and/or secretion
of TGF-s. We have directly tested this hypothesis in MCF-7 and T47D
breast carcinoma cells, which exhibit enhanced secretion of TGF-
bioactivity upon treatment with the antiestrogen tamoxifen. All this
secreted TGF-
activity was in a latent form requiring acid
activation in vitro for it to be detected. This may reflect
the inability of the radioreceptor assay to detect TGF-
s already
utilized by the cells as well as to estimate in situ
activation of TGF-
s at 37 °C over a more prolonged time and in
the presence of a potential target cell. Supporting the latter
possibility, medium conditioned by MCF-7 cells in the presence of
tamoxifen inhibits the growth of cocultured tamoxifen-insensitive MDA-231 breast cancer cells. This inhibition by MCF-7 cell medium was
reversed by anti-TGF-
antibodies (22).
MCF-7 cells but not the T47D line express TGF-RII mRNA (32).
Although the growth inhibitory response of both cell lines to high
concentrations of exogenous TGF-
1 and TGF-
2 is minor (36, 37),
this does not rule out a potential response to lower concentrations of
endogenous TGF-
s. The absence of TGF-
RII mRNA and protein,
presumably indispensable for TGF-
cellular responses, in
tamoxifen-sensitive T47D cells argues per se against
TGF-
's involvement in antiestrogen response. However, these cells
bind TGF-
1 (37, Fig. 1) and, in response to the progestin analog
gestodene, secrete 90-fold higher levels of TGF-
1 and -
2 proteins
and become growth-arrested (28). This inhibitory effect of gestodene in T47D cells is partially reversed by a polyclonal TGF-
antiserum, suggesting these cells are perhaps responsive to autocrine TGF-
s despite lacking TGF-
RII.
We first used antibodies that neutralize mature TGF-s in an attempt
to block antiestrogen action. This approach has been shown to partially
block retinoic acid-mediated inhibition of keratinocytes by
counteracting the autocrine action of TGF-
2 (42). In our study,
blockade of all three mammalian TGF-
isoforms with different
monoclonal antibodies did not alter tamoxifen-induced cell cycle arrest
or inhibition of MCF-7 and T47D colony growth. Furthermore,
transfection of TGF-
RII into MCF-7 cells did not enhance their
sensitivity to tamoxifen, even though the antiestrogen did enhance the
secretion of TGF-
activity and binding by these cells. These results
also argue that, at pharmacologically achievable concentrations,
tamoxifen does not require the contribution of autocrine TGF-
to
induce growth inhibition.
Treatment with tamoxifen increased ligand binding by all three TGF-
receptor types. This may argue against an autocrine interaction between
secreted TGF-
s and endogenous receptors as shown with mouse
keratinocytes by Glick et al. (42). Upon terminal
differentiation, these cells exhibit a 10-20-fold increase in TGF-
2
mRNA and peptide with simultaneous down-regulation of type I and II
TGF-
receptors available for affinity cross-linking; a mild acid
wash significantly increased the number of receptor sites in
differentiated keratinocytes suggesting masking of TGF-
receptors by
endogenous ligand (43). On the other hand, other steroid molecules can
up-regulate TGF-
receptors at the protein and/or mRNA level
despite a simultaneous increase in expression/secretion of TGF-
ligands (44). Further work is needed to study the mechanism(s) by which
tamoxifen up-regulates TGF-
receptors in MCF-7 and T47D cells.
Preliminary experiments with MCF-7 cells, however, failed to show an
increase in steady-state TGF-
RII mRNA levels after a 24-h
incubation with 1 µM
tamoxifen,4 arguing against a
transcriptional effect to explain the result with MCF-7/RII cells (Fig.
3B).
TGF- activation can occur locally within the cell surface of target
cells (45, 46). Neutralizing anti-TGF-
antibodies may not be able to
block TGF-
activity to a threshold required for the reversal of
growth inhibitory signals or alter a ligand-independent direct effect
of tamoxifen on TGF-
RII signaling. Therefore, we examined the effect
of a kinase negative truncated TGF-
RII (
RII) on tamoxifen
response in early passage MCF-7 cells. These cells are more sensitive
than late passage MCF-7 cells to exogenous TGF-
1 and exhibit
detectable levels of TGF-
RII at the cell surface thus providing an
appropriate model to test directly both TGF-
1 and antiestrogen
response. TGF-
1 responses were abrogated by the dominant negative
RII mutant.2 However, tamoxifen-mediated cell cycle
arrest, suppression of Rb phosphorylation, and antiproliferative
effects in these MCF-7 cells were identical with or without endogenous
TGF-
RII signaling, disproving any major role for autocrine TGF-
s
on the response to antiestrogens.
Once a dissociation between TGF-s- and tamoxifen-mediated growth
inhibition was established, we studied whether they independently suppressed Rb phosphorylation by similar mechanisms in the MCF-7/
RII cells. The pure antiestrogen ICI182780 and TGF-
1 induce p21 and p27,
which, by complexing with the cyclin E-Cdk2 complex, prevent Rb
phosphorylation and hence progression beyond the G1 phase
of the cell cycle (7, 39-41, 47). Exogenous TGF-
1 but not tamoxifen induced p21 as well as association of p21 with Cdk2. These responses were abrogated by the
RII mutant receptor, supporting the need of
intact TGF-
RII signaling to elicit ligand-mediated effects on the
Cdk2 inhibitor p21. Neither tamoxifen nor TGF-
1 induced p27 in
MCF-
RII cells. In addition to the dissociation between both growth
inhibitory pathways at a cellular level, these data with p21 further
suggest that TGF-
s and tamoxifen suppress Rb phosphorylation by
independent molecular mechanisms.
These data, generated with cell-autonomous experimental systems, do not
rule out a possible role for antiestrogen-induced TGF-s in the
anti-tumor response to tamoxifen in clinical breast carcinoma by a
paracrine/endocrine mechanism. Conflicting data have been published on
this topic. Butta et al. reported that 3 months of tamoxifen
therapy resulted in ER-independent enhanced TGF-
1 staining around
stromal fibroblasts in breast tumor biopsies (48). The correlation
between antiestrogen-induced enhancement of peritumoral TGF-
1
protein and a clinical response was not reported in this study. In two
other studies, a rise in the circulating level of TGF-
2 (49) or in
the tumor levels of TGF-
2 mRNA (50) correlated with a clinical
response to antiestrogens, suggesting up-regulation of TGF-
s is a
surrogate marker or epiphenomenon of an anti-tumor effect. On the other
hand, a more recent immunohistochemical study in 19 patients failed to
show alterations in TGF-
1 staining with intervening tamoxifen
therapy, despite a >50% clinical response rate (51). Transfection of
MCF-7 cells with a TGF-
1 expression vector does not alter tamoxifen
sensitivity (52). Finally, breast tumors unresponsive to tamoxifen,
when rebiopsied, expressed significantly higher levels of TGF-
1
mRNA than clinically responsive tumors (53). A causal association
between ligand overexpression and the antiestrogen-resistant phenotype,
if any, would require additional mechanistic studies.
In any event, the data presented strongly argue against a significant
involvement of TGF- ligands and receptor signaling on the growth
inhibition of human breast carcinoma cells by antiestrogens. Prospective epidemiologic studies will likely address whether treatment-induced up-regulation of TGF-
s expression in tumors in situ can be used as a marker of response (or lack of
response) to antiestrogens. Although it is still possible that
autocrine/paracrine TGF-
s can be involved in antiestrogen response
in some mammary carcinomas, the effect of TGF-
s on the host's
immune system and on tumor's stroma, cell adhesion, and angiogenesis
(reviewed in Ref. 54) can easily mask the net contribution of this
putative autocrine pathway to breast tumor cell viability and
progression by indirectly favoring breast tumor maintenance.