Blockade of Transforming Growth Factor-beta Signaling Does Not Abrogate Antiestrogen-induced Growth Inhibition of Human Breast Carcinoma Cells*

(Received for publication, October 28, 1996, and in revised form, December 30, 1996)

Katri M. Koli Dagger §, Timothy T. Ramsey Dagger , Yong Ko , Teresa C. Dugger Dagger , Michael G. Brattain and Carlos L. Arteaga Dagger §par **Dagger Dagger

From the Departments of Dagger  Medicine and par  Cell Biology, Vanderbilt University School of Medicine, § Vanderbilt Cancer Center, and ** Department of Veteran Affairs Medical Center, Nashville, Tennessee 37232 and the  Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43699

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

We have studied the role of autocrine transforming growth factor-beta (TGF-beta ) signaling on antiestrogen-mediated growth inhibition of hormone-dependent T47D and MCF-7 human breast carcinoma cells. Tamoxifen treatment increased the secretion of TGF-beta activity into serum-free cell medium and the cellular content of affinity cross-linked type I and III TGF-beta receptors in both cell lines. Anti-pan-TGF-beta 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-beta receptors at the cell surface and exquisite sensitivity to exogenous TGF-beta 1, were transfected with a tetracycline-controllable dominant-negative TGF-beta RII (Delta RII) construct. Although the TGF-beta 1 response was blocked by removal of tetracycline in MCF-7/Delta 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-beta 1 treatment up-regulated the Cdk inhibitor p21 and induced its association with Cdk2 in MCF-7 cells; these responses were blocked by the Delta RII transgene product. In MCF-7 cells with a functional TGF-beta 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-beta 1 unresponsive MCF-7 cells with high levels of TGF-beta RII restored TGF-beta 1-induced growth inhibition but did not enhance tamoxifen response in culture. Taken together these data strongly argue against any role for TGF-beta signaling on tamoxifen-mediated growth inhibition of hormone-dependent breast cancer cells.


INTRODUCTION

Transforming growth factor-beta s (TGF-beta 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-beta is its ability to inhibit cell proliferation (4). Three different mammalian TGF-beta isoforms (TGF-beta 1, -beta 2, and -beta 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-beta 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-beta and its receptor molecules are expressed ubiquitously by normal and transformed cells.

Both signaling receptors seem to be needed for TGF-beta responsiveness (8), and type II receptor expression correlates with the anti-proliferative activity of TGF-beta (9, 10). TGF-beta arrests cell growth in the G1 phase of the cell cycle and most probably affects multiple signaling pathways (11). TGF-beta 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-beta contributes to the accumulation of hypophosphorylated pRb (13). In several cell lines, TGF-beta 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.

All three TGF-beta 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-beta administrated by slow release pellets or tissue-specific expression of active TGF-beta 1 in the mammary gland of transgenic mice leads to ductal hypoplasia and suppression of ductal branching (16, 18, 19).

Normal and tumorigenic human breast epithelial cells in culture express TGF-beta 1 mRNA and secrete TGF-beta receptor binding activity into their medium (20, 21). Published data support the notion that endogenous TGF-beta s function as autocrine growth regulators of breast cancer cell proliferation (22, 23). Antibodies that neutralize mature TGF-beta 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-beta 2 and -beta 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-beta 1 mRNA expression or increased secretion of TGF-beta bioactivity or protein synthesis without associated mRNA changes (22, 27, 28), thus leading to the hypothesis that autocrine TGF-beta signaling contributed to antiestrogen's actions. Some reports, however, argue against TGF-beta s' role in the growth inhibitory response to antiestrogens. MCF-7 and T47D breast cancer cells can exhibit resistance to TGF-beta 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-beta RII and hence response to exogenous TGF-beta 1, remain sensitive to the cytostatic effect of tamoxifen (31, 32). By using anti-TGF-beta neutralizing antibodies as well as dominant negative TGF-beta RII constructs in estrogen-dependent, tamoxifen-sensitive human breast cancer cells, we have formally tested in this study the role of endogenous TGF-beta signaling on the cellular response to antiestrogens in breast carcinoma.


MATERIALS AND METHODS

Cell Lines and Antibodies

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-beta 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-beta receptor (MCF-7/Delta RII)2 were cultured in IMEM containing 10% FCS and 500 µg/ml G418 (Life Technologies, Inc.). The alpha -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-beta 1 and have been characterized previously (34). The 12H5 IgG2 is devoid of TGF-beta neutralizing activity, while the 4A11 IgG1 and the 2G7 IgG2 neutralize the growth inhibitory activity of TGF-beta 1 and TGF-beta 1, -beta 2, and -beta 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-beta RII.

Collection of Cell-conditioned Medium (CM) and TGF-beta Radioreceptor Assay

Secreted TGF-beta 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-beta 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-beta 1 (specific activity 173 µCi/µg; DuPont NEN) with or without variable volumes of CM for 4 h at 4 °C. Human recombinant TGF-beta 1 (Genentech) was used to generate a standard curve from which the receptor binding activity in CM was calculated. In this assay, TGF-beta 1 and TGF-beta 2 are equipotent in displacing 125I-TGF-beta 1 binding (35). Addition of 1 µM tamoxifen directly to the radioreceptor assay has no effect on TGF-beta 1 binding by AKR-2B cells.3

125I-TGF-beta Binding and Affinity Cross-linking

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-beta 1 with or without 100-fold excess unlabeled TGF-beta 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-beta 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-beta 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-beta 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.

Analysis of Cell Cycle Distribution

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).

Cell Proliferation Assays

Cells (2 × 104/well) were plated in 24-well dishes in regular growth medium. The following day, 1 µM tamoxifen or TGF-beta 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-beta 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).

Cdk2 Immunoprecipitation and Immunoblot Analysis of pRb, p21, and p27

Adherent monolayers were treated with tamoxifen or TGF-beta 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 alpha -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.


RESULTS

Tamoxifen Increases Secreted TGF-beta Bioactivity and TGF-beta Binding in MCF-7 and T47D Cells

We first examined the modulation of TGF-beta 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-beta activity was measured in a 125I-TGF-beta 1 radioreceptor assay. In the absence of acid activation in vitro, TGF-beta activity was below the detection limit of the binding assay, indicating that the majority of the secreted TGF-beta was in a latent form. Tamoxifen induced increases of approximately 30- and 4-fold in the secretion of acid-activable TGF-beta activity in MCF-7 and T47D cultures, respectively (Table I).

Table I.

Tamoxifen increases the secretion of TGF-beta activity by MCF-7 and T47D cells

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-beta activity in CM was measured in a 125I-TGF-beta 1 radioreceptor assay with AKR-2B mouse fibroblasts as indicator cells. TGF-beta 1 equivalents of receptor binding activity in CM were extrapolated from a competition standard curve with recombinant unlabeled TGF-beta 1 and standardized to cell number.


Treatment TGF-beta 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-beta 1 equivalents/106 cells/24 h.

To test whether endogenous TGF-beta ligands in response to the antiestrogen were masking endogenous TGF-beta receptors, we examined the effect of tamoxifen on 125I-TGF-beta 1 binding and the cellular content of affinity cross-linked TGF-beta receptors. Both MCF-7 and T47D cells exhibit type I and type III TGF-beta receptors at the cell surface, whereas TGF-beta RII was undetectable (Fig. 1). To enhance the sensitivity of the assay, we labeled 5 × 107 cells with 10 ng/ml 125I-TGF-beta 1 in a 1-ml suspension followed by cross-linking and immunoprecipitation with TGF-beta 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-beta 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-beta 1 binding in control and tamoxifen-treated cells,3 arguing against significant masking of TGF-beta binding sites by autocrine ligands in the presence or absence of the antiestrogen.


Fig. 1. Tamoxifen-induced up-regulation of TGF-beta binding in MCF-7 and T47D cells. Subconfluent cell monolayers were treated for 48 h with 0.1% ethanol (-) or 1 µM tamoxifen (Tam, +) and then labeled with 1 ng/ml 125I-TGF-beta 1 in the presence of absence of 100-fold excess unlabeled TGF-beta 1 for 4 h at 4 °C. Labeled receptors were cross-linked with 50 µM disuccinimidyl suberate as described under "Materials and Methods," solubilized, and resolved by 5-7% gradient SDS-PAGE. The TGF-beta type I and III receptors are indicated by the arrows. Molecular size markers in kDa are indicated to the left of each panel.
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Neutralizing Anti-TGF-beta Antibodies Do Not Block Tamoxifen Growth Inhibitory Action

To determine whether the enhanced TGF-beta secreted activity was mediating tamoxifen actions, we performed studies with anti-TGF-beta antibodies. These antibodies have been shown to stimulate the proliferation of breast tumor cells with an operative autocrine TGF-beta pathway by neutralizing endogenous mature TGF-beta 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-beta 2G7 or the anti-TGF-beta 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-beta antibodies (Fig. 2B). By themselves, the IgGs utilized had no growth effects on either cell line (data not shown).


Fig. 2. Anti-TGF-beta neutralizing antibodies do not abrogate tamoxifen-mediated cytostasis. A, cells were treated in regular growth medium with 0.1% ethanol or 1 µM tamoxifen plus the indicated IgGs (100 µg/ml) for 48 h. After this incubation, the monolayers were trypsinized, cell nuclei labeled with PI, and DNA histograms analyzed by flow cytometry. B, a suspension of 3 × 104 MCF-7 or T47D cells was plated in a soft agarose colony forming assay in the presence of 0.1% ethanol or 1 µM tamoxifen (Tam) plus the indicated IgGs (100 µg/ml) as described under "Materials and Methods." Colonies measuring >=  50 µM were counted 10 days later. Each data point represents the mean ± S.D. of three dishes.
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High Expression of Functional TGF-beta RII Does Not Enhance Tamoxifen Action in MCF-7 Cells

Transfection with high levels of a tetracycline-repressible TGF-beta RII expression vector into MCF-7 cells (MCF-7/RII cells, Ref. 9) restores sensitivity to growth inhibition by exogenous TGF-beta . Therefore, in these cells, we tested whether up-regulation of an operative TGF-beta 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-beta RII. Similar to its effect on endogenous TGF-beta RI and TGF-beta RIII in MCF-7 and T47D cells (Fig. 1), tamoxifen also increased the cellular content of TGF-beta 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-beta 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-beta RII were not useful.


Fig. 3. A, enhanced expression of TGF-beta RII does not enhance tamoxifen action in MCF-7 cells. 2 × 104 MCF-7/RII cells were seeded in 24-well plates in regular growth medium with or without 0.1 µM tetracycline (tet). The following day 0.1% ethanol or 1 µM tamoxifen were added. Cell proliferation was determined 4 days later in a Coulter counter. Each bar represents the mean ± S.D. of triplicate wells. B, tamoxifen-mediated up-regulation of TGF-beta binding in MCF-7/RII cells. Subconfluent monolayers of MCF-7/RII cells were incubated for 48 h with 0.1% ethanol (-) or 1 µM tamoxifen (+) in the presence or absence of 0.1 µM tetracycline. Cells were then labeled with 125I-TGF-beta 1 and ligand-binding proteins cross-linked and visualized as indicated under "Materials and Methods" and the legend of Fig. 1.
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Expression of a Dominant Negative TGF-beta RII Does Not Block Cellular Responses to Anti-estrogens in MCF-7 Cells

Since the neutralizing TGF-beta antibodies may not be effective in blocking a potential ligand/receptor intracellular coupling or a direct ligand-independent effect of tamoxifen on TGF-beta RII, we used a dominant negative approach to block TGF-beta 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-beta RII protein at the cell surface and are markedly growth inhibited by exogenous TGF-beta s (36). Expression of a tetracycline-repressible dominant negative type II TGF-beta receptor in these cells (MCF-7/Delta RII) blocks cellular responses to exogenous TGF-beta 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 Delta 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).


Fig. 4. A dominant negative type II TGF-beta receptor (Delta RII) does not block tamoxifen-induced growth inhibition of MCF-7/Delta RII cells. A, 2 × 104 MCF-7/Delta RII cells were seeded in 24-well plates in regular growth medium with or without 0.1 µM tetracycline (tet). The following day 0.1% ethanol (Ctrl) or 1 µM tamoxifen (Tam) were added. Cell counts were determined 4 days later in Coulter counter. Each bar represents the mean ± S.D. of triplicate wells. B, a suspension of 3 × 104 MCF-7/Delta RII cells was plated in a soft agarose colony forming assay in the presence or absence of 0.1 µM tetracycline. The following day 0.1% ethanol or 1 µM tamoxifen (in 0.1 ml of sterile PBS) were added to the surface of top agarose layer. Colonies measuring >= 50 µM were counted 10 days later. Each data point represents the mean ± S.D. of three dishes.
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Both TGF-beta 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 Delta RII mutant on tamoxifen-mediated cell cycle arrest and Rb phosphorylation in MCF-7/Delta 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/Delta RII cells under conditions in which the Delta RII mutant type II receptor was expressed or not (Fig. 6) further arguing against any role for endogenous TGF-beta signaling on antiestrogen-mediated G1 arrest.


Fig. 5. Tamoxifen-induced G1 arrest is not blocked by the Delta RII mutant receptor. MCF-7/Delta RII cells were seeded in regular growth medium with or without 0.1 µM tetracyclin. The following day 0.1% ethanol (-) or 1 µM tamoxifen (+) were added for 72 h. After this incubation, the monolayers were trypsinized, cell nuclei labeled with PI, and DNA histograms generated in a FACScan flow cytometer. A, DNA histograms of PI-labeled MCF-7/Delta RII nuclei. B, table of cell cycle distribution of MCF-7/Delta RII cells under different treatment conditions based on analysis of 50,000 PI-labeled nuclei as indicated under "Materials and Methods."
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Fig. 6. Suppression of Rb phosphorylation by antiestrogens is not blocked by the Delta RII dominant negative receptor. MCF-7/Delta RII were plated in regular growth medium with or without 0.1 µM tetracycline. The following day 0.1% ethanol or 1 µM tamoxifen were added for 24 h. Cells were washed twice with ice-cold PBS and solubilized with EBC buffer as described under "Materials and Methods." 150 µg of protein (BCA method) were then subjected to 7% SDS-PAGE followed by an Rb immunoblot procedure. Molecular size markers in kDa are indicated on the left.
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TGF-beta 1 but Not Tamoxifen Induces p21WAF1/CIP1 in MCF-7 Cells

TGF-beta 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-beta 1 and tamoxifen suppressed Rb phosphorylation by inducing p21 in MCF-7/Delta RII cells. An overnight incubation with 1 ng/ml TGF-beta 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-beta 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-beta s. Prolongation of tamoxifen treatment to 72 h still did not induce p21 protein. A 24-h treatment of MCF-7/Delta RII cells with 1 µM tamoxifen or 1 ng/ml TGF-beta 1 in the presence or absence of tetracycline, did not induce the Cdk inhibitor p27 as measured by immunoblot analysis (data not shown).


Fig. 7. TGF-beta 1 but not tamoxifen induces p21 and p21-Cdk2 association. MCF-7/Delta RII cells were plated as in Fig. 6 in the presence or absence of tetracycline and the following day treated for 24 h with 0.1% ethanol (ctl), 1 µM tamoxifen (Tam), or 1 ng/ml recombinant TGF-beta 1. After this incubation, total cell lysates were prepared as indicated under "Materials and Methods" and 150 µg of protein/treatment condition subjected to p21 immunoblotting (top). To assess association of p21 with Cdk2, 300 µg of protein/treatment condition were precipitated with 2 µg of a Cdk2 antiserum (Cdk2ip). Coprecipitated p21 was assessed by immunoblot analysis of immune complexes as indicated under "Materials and Methods" (bottom).
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DISCUSSION

It is proposed that antiestrogens induce growth inhibition of human breast tumor cells by up-regulating expression and/or secretion of TGF-beta s. We have directly tested this hypothesis in MCF-7 and T47D breast carcinoma cells, which exhibit enhanced secretion of TGF-beta bioactivity upon treatment with the antiestrogen tamoxifen. All this secreted TGF-beta 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-beta s already utilized by the cells as well as to estimate in situ activation of TGF-beta 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-beta antibodies (22).

MCF-7 cells but not the T47D line express TGF-beta RII mRNA (32). Although the growth inhibitory response of both cell lines to high concentrations of exogenous TGF-beta 1 and TGF-beta 2 is minor (36, 37), this does not rule out a potential response to lower concentrations of endogenous TGF-beta s. The absence of TGF-beta RII mRNA and protein, presumably indispensable for TGF-beta cellular responses, in tamoxifen-sensitive T47D cells argues per se against TGF-beta 's involvement in antiestrogen response. However, these cells bind TGF-beta 1 (37, Fig. 1) and, in response to the progestin analog gestodene, secrete 90-fold higher levels of TGF-beta 1 and -beta 2 proteins and become growth-arrested (28). This inhibitory effect of gestodene in T47D cells is partially reversed by a polyclonal TGF-beta antiserum, suggesting these cells are perhaps responsive to autocrine TGF-beta s despite lacking TGF-beta RII.

We first used antibodies that neutralize mature TGF-beta 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-beta 2 (42). In our study, blockade of all three mammalian TGF-beta 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-beta RII into MCF-7 cells did not enhance their sensitivity to tamoxifen, even though the antiestrogen did enhance the secretion of TGF-beta activity and binding by these cells. These results also argue that, at pharmacologically achievable concentrations, tamoxifen does not require the contribution of autocrine TGF-beta to induce growth inhibition.

Treatment with tamoxifen increased ligand binding by all three TGF-beta receptor types. This may argue against an autocrine interaction between secreted TGF-beta 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-beta 2 mRNA and peptide with simultaneous down-regulation of type I and II TGF-beta receptors available for affinity cross-linking; a mild acid wash significantly increased the number of receptor sites in differentiated keratinocytes suggesting masking of TGF-beta receptors by endogenous ligand (43). On the other hand, other steroid molecules can up-regulate TGF-beta receptors at the protein and/or mRNA level despite a simultaneous increase in expression/secretion of TGF-beta ligands (44). Further work is needed to study the mechanism(s) by which tamoxifen up-regulates TGF-beta receptors in MCF-7 and T47D cells. Preliminary experiments with MCF-7 cells, however, failed to show an increase in steady-state TGF-beta 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-beta activation can occur locally within the cell surface of target cells (45, 46). Neutralizing anti-TGF-beta antibodies may not be able to block TGF-beta activity to a threshold required for the reversal of growth inhibitory signals or alter a ligand-independent direct effect of tamoxifen on TGF-beta RII signaling. Therefore, we examined the effect of a kinase negative truncated TGF-beta RII (Delta RII) on tamoxifen response in early passage MCF-7 cells. These cells are more sensitive than late passage MCF-7 cells to exogenous TGF-beta 1 and exhibit detectable levels of TGF-beta RII at the cell surface thus providing an appropriate model to test directly both TGF-beta 1 and antiestrogen response. TGF-beta 1 responses were abrogated by the dominant negative Delta 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-beta RII signaling, disproving any major role for autocrine TGF-beta s on the response to antiestrogens.

Once a dissociation between TGF-beta s- and tamoxifen-mediated growth inhibition was established, we studied whether they independently suppressed Rb phosphorylation by similar mechanisms in the MCF-7/Delta RII cells. The pure antiestrogen ICI182780 and TGF-beta 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-beta 1 but not tamoxifen induced p21 as well as association of p21 with Cdk2. These responses were abrogated by the Delta RII mutant receptor, supporting the need of intact TGF-beta RII signaling to elicit ligand-mediated effects on the Cdk2 inhibitor p21. Neither tamoxifen nor TGF-beta 1 induced p27 in MCF-Delta RII cells. In addition to the dissociation between both growth inhibitory pathways at a cellular level, these data with p21 further suggest that TGF-beta 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-beta 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-beta 1 staining around stromal fibroblasts in breast tumor biopsies (48). The correlation between antiestrogen-induced enhancement of peritumoral TGF-beta 1 protein and a clinical response was not reported in this study. In two other studies, a rise in the circulating level of TGF-beta 2 (49) or in the tumor levels of TGF-beta 2 mRNA (50) correlated with a clinical response to antiestrogens, suggesting up-regulation of TGF-beta 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-beta 1 staining with intervening tamoxifen therapy, despite a >50% clinical response rate (51). Transfection of MCF-7 cells with a TGF-beta 1 expression vector does not alter tamoxifen sensitivity (52). Finally, breast tumors unresponsive to tamoxifen, when rebiopsied, expressed significantly higher levels of TGF-beta 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-beta 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-beta 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-beta s can be involved in antiestrogen response in some mammary carcinomas, the effect of TGF-beta 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.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants CA62212 (to C. L. A.), CA38173 (to M. G. B.), and CA54807 (to M. G. B.); Merit Review and Clinical Investigator grants from the Department of Veteran Affairs (to C. L. A.); and support from the T. J. Martell Foundation (to C. L. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger Dagger    To whom correspondence should be addressed: Div. of Medical Oncology, Vanderbilt University, 1161 22nd Ave. S., 1956 TVC, Nashville, TN 37232-5536. Tel.: 615-936-1919; Fax: 615-343-7602; E-mail: carlos.arteaga{at}mcmail.vanderbilt.edu.
1   The abbreviations used are: TGF, transforming growth factor; TGF-beta RII, type II TGF-beta receptor; Rb, retinoblastoma protein; Cdk, cyclin-dependent kinase; IMEM, improved minimal essential medium; FCS, fetal calf serum; CM, conditioned medium; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; BCA, bicinchoninic acid; ER, estrogen receptor; PI, propidium iodide.
2   Y. Ko, K. Koli, W. Li, J. K. V. Willson, M. G. Brattain, and C. L. Arteaga, submitted for publication.
3   C. L. Arteaga and T. C. Dugger, unpublished results.
4   A. E. Gorska and C. L. Arteaga, unpublished results.

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