©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transforming Growth Factor- -induced Cell Growth Inhibition in Human Breast Cancer Cells Is Mediated through Insulin-like Growth Factor-binding Protein-3 Action (*)

Youngman Oh (§) , Hermann L. Müller (¶) , Lilly Ng , Ron G. Rosenfeld

From the (1) Department of Pediatrics, School of Medicine, Oregon Health Sciences University, Portland, Oregon 97201-3042

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Most estrogen receptor-negative breast cancer cells, including Hs578T cells, express mRNAs encoding insulin-like growth factor-binding protein (IGFBP)-3, as well as transforming growth factor (TGF)- receptors. Our previous studies (Oh, Y., Muller, H. L., Lamson, G., and Rosenfeld, R. G.(1993) J. Biol. Chem. 268, 14964-14971; Oh, Y., Muller, H. L., Pham, H. M., and Rosenfeld, R. G.(1993) J. Biol. Chem. 268, 26045-26048) have demonstrated a significant inhibitory effect of exogenous IGFBP-3 on Hs578T cell growth and existence of IGFBP-3-specific receptors that may mediate those direct inhibitory effect of IGFBP-3. TGF- is also a potent growth inhibitor in human breast cancer cells in vitro and regulates IGFBP-3 production in different cell systems, suggesting that IGFBP-3 is a major anti-proliferative factor and a key element for TGF--induced growth inhibition in human breast cancer cells. In support of this hypothesis, we have demonstrated using Hs578T cells that: 1) TGF- stimulates IGFBP-3 gene expression and production prior to its inhibition of cell growth, 2) treatment with an IGFBP-3 antisense oligodeoxynucleotide selectively inhibits TGF--induced IGFBP-3 synthesis and cell growth inhibition, and 3) treatment with IGF-II and IGF-II analogs diminish TGF- effects by blocking TGF--induced binding of IGFBP-3 to the cell surface. These findings suggest that IGFBP-3 is a major anti-proliferative factor and a key element in TGF--induced growth inhibition in human breast cancer cells.


INTRODUCTION

The insulin-like growth factor-binding proteins (IGFBPs)() bind specifically to the insulin-like growth factors (IGF-I and IGF-II) and modulate the mitogenic effects of IGFs in various systems (1) . IGFs are the most potent mitogens, apart from estrogens, for human breast cancer cells (2) and exert their mitogenic effect mainly through the IGF-I receptor, which possesses an intrinsic ligand-activated tyrosine kinase activity (3). Recently, our studies have demonstrated a significant inhibitory effect of exogenous IGFBP-3 on the growth of Hs578T cells (4) and IGFBP-3-transfected mouse Balb/c fibroblast cells (5) . Furthermore, we have demonstrated the existence of IGFBP-3-specific receptors that may mediate the direct inhibitory effect of IGFBP-3 on Hs578T cells, and we have shown that IGFBP-3 can inhibit cell proliferation by itself, even in the absence of IGFs (6) .

The growth of human breast cancer cells can be inhibited in vitro by several factors, including TGF- (7) , retinoic acid (8), and anti-estrogens (9, 10) . It has been reported that these anti-proliferative factors stimulate expression of IGFBP-3 in various cell systems (8, 11, 12, 13) . In particular, TGF- is expressed by most human breast cancer cells (7, 14) and by stromal fibroblasts, regardless of whether the fibroblasts originate from normal or malignant cells, indicating that TGF- is part of a tightly balanced negative growth factor system in vivo(15) . The exact mechanisms by which TGF- inhibits breast cancer cell proliferation are not well understood. In this study, we demonstrate that the inhibitory effect of TGF- on Hs578T cell growth is mediated, at least in part, by enhanced expression of IGFBP-3.


MATERIALS AND METHODS

Peptides and Proteins

Recombinant human IGF-II was provided by Lilly. [Leu]IGF-II and [Gln,Ala,Tyr,Leu,Leu]IGF-II, synthetic IGF-II analogs, were synthesized as described previously (16). Recombinant TGF-2 was the generous gift of Celtrix, Inc. (Richmond, CA). Iodination of IGF-II to a specific activity of 350-500 µCi/µg was performed by a modification of the chloramine-T technique.

IGFBP-3 Antisense Phosphorothioate Oligodeoxynucleotide (ODN)

The IGFBP-3 antisense and sense ODNs used in this experiment were prepared by OLIGOS Etc., Inc. (Guilford, CT). The IGFBP-3 antisense ODN was complementary to 20 nucleotides that encode the N terminus of human IGFBP-3 (17) and had the sequence 5`-CAT GAC GCC TGC AAC CGG GG-3` (positions 2021-2040); the sequence of the IGFBP-3 sense ODN was 5`-CCC CGG TTG CAG GCG TCA TG-3`. A search of GenBank revealed no other matching sequences.

Cell Cultures

Hs578T human breast cancer cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/liter glucose, 110 mg/liter sodium pyruvate, and 10% fetal bovine serum as described previously (4) .

Preparation of Conditioned Medium (CM)

Cells were grown until 95% confluent in serum-containing media and then switched for 12 h to serum-free DMEM. Medium was aspirated again, and cells were maintained in serum-free DMEM with or without TGF-2 or IGFBP-3 ODN for various times indicated in the text. Conditioned media were collected and centrifuged at 1000 g for 10 min to remove cell debris. The harvested CM from triplicate wells within each experiment were pooled and stored at -70 °C until assay.

Western Ligand Blots

Proteins from CM samples were size-fractionated by SDS-polyacrylamide gel electrophoresis under non-reducing conditions and electroblotted onto nitrocellulose filters (0.45-µm pore size) using a BioTrans unit. Filters were incubated overnight with 1.5 10 cpm of I-labeled IGF-II, washed, dried, and exposed to film.

Monolayer Cell Replication Assay

Cells were grown in 12-well plates until 60% confluent (0.2 10 cells/well) and changed to serum-free media for 12 h, and then maintained in serum-free DMEM containing 0.25% bovine serum albumin in the presence or absence of different concentrations of TGF-2 or IGFBP-3 ODN for various time points as indicated in the text. Cells were then gently detached from plates by phosphate-buffered saline/EDTA and cell number counted using either a hematocytometer or a Coulter Counter (Coulter, Ltd., Beds., United Kingdom). In experiments investigating the effect of IGFBP-3 ODN and IGF peptide on the growth-inhibitory action of TGF-2, cells were incubated with 5 ng/ml TGF-2 in the presence of various concentrations of IGFBP-3 ODN, IGF-II, or IGF-II analogs for 5 days.

Northern Blots

Total cellular RNA was isolated from cells by a modification of the guanidinium isothiocyanate-LiCl precipitation method (18) . Sample RNA was size-fractionated on 1.2% agarose-formaldehyde gels and then transferred to nitrocellulose. Blotted RNAs were hybridized sequentially with cDNA probes labeled with [P]dCTP by random priming (Pharmacia, Uppsala, Sweden); bands were visualized by autoradiography after exposure to XAR film with intensifying screens at -70 °C. RNA ladder size markers (Life Technologies, Inc.) were used to obtain size estimates of specific transcripts. The human IGFBP-3 probe was a 1082-base pair EcoRI-PvuII fragment of the human IGFBP-3 cDNA that includes the entire coding region sequence (cDNA kindly provided by Dr. W. Wood, Genentech, South San Francisco, CA). Band densities were analyzed using the area under the curve, as calculated by a LKB densitometer. The relative density of the bands corresponding to IGFBP-3 on autoradiography were expressed as absorption units per millimeter.

Statistical Analysis

Data were analyzed with a two-tailed Student's t test, using the software program Statview (Abacus Concepts, Inc.)


RESULTS

We first investigated whether treatment with TGF-2 would stimulate expression of IGFBP-3 by Hs578T cells. As in previous studies (7), 5 days of treatment with 1 ng/ml TGF-2 inhibited cell growth by 40% relative to control cells (p < 0.05), as shown in Fig. 1A. A time-course experiment with 5 ng/ml TGF-2 showed that the inhibitory effect of TGF-2 on monolayer growth occurs during days 4-6 of treatment, with maximal inhibition at day 5 (Fig. 1B). We next examined the effect of TGF-2 on IGFBP-3 mRNA levels and the levels of secreted protein. Hs578T cells secrete IGFBP-3 (shown as 41-kilodalton (kDa) and 39-kDa IGF-binding species) and IGFBP-4 (24-kDa species) as major IGFBPs. Treatment with 5.0 ng/ml TGF-2 for 2 days increased IGFBP-3 levels in conditioned media by 3.0-fold, whereas CM IGFBP-4 levels were not changed significantly by the same concentrations of TGF-2 (Fig. 2A). As reported previously, IGFBP-3-specific proteases are major regulators of secreted and cell surface-associated IGFBP-3 levels (19) . To determine if the effect of TGF-2 on IGFBP-3 resulted from changes in IGFBP-3 protease activity, we performed IGFBP-3 protease assays as described previously (20) . No IGFBP-3 protease activity was detectable either in the presence or absence of TGF-2 (data not shown).


Figure 1: Effect of TGF-2 on Hs578T cell growth. A, dose response of TGF-2 effect. Hs578T cells were grown until 60% confluent (0.2 10 cells/well) and then changed to serum-free media containing 0.25% bovine serum albumin with various concentrations of TGF-2. After 5 days of treatment, cell numbers were counted with a Coulter Z1 counter (Coulter, Ltd). Results represent means ± S.E. of two separate experiments performed in triplicate. *, two-tailed t test; p < 0.05. B, time course of growth inhibition with 5 ng/ml TGF-2. Experiments are similar to those shown in A. The data are expressed as the percent of control cell numbers at each time point.




Figure 2: Effect of TGF-2 on IGFBP-3 production in Hs578T cells. A, IGFBP-3 concentrations in CM from TGF-2-treated cells. Representative Western ligand blot of CM from cells incubated in DMEM alone (lane1) or with 0.05-5.0 ng/ml TGF-2 (lanes 2-6) for 2 days. Arepresentative gel from one of the two experiments is shown. B, effect of TGF-2 on IGFBP-3 mRNA levels. Cells were grown until 90% confluent and incubated with serum-free media containing 1 or 5 ng/ml TGF-2 for 8 h. Total RNA was extracted and analyzed by Northern blot. A representative blot from two separate experiments is shown.



Northern blot analysis of total RNA from Hs578T cells revealed a single, 2.6-kilobase mRNA species that hybridized to a human IGFBP-3 cDNA probe. Treatment with 5 ng/ml TGF-2 for 8 h resulted in a 2.0-fold increase in IGFBP-3 mRNA, after correction for -actin mRNA (Fig. 2B). Similar results were obtained with TGF-1 (data not shown). The observed increase in IGFBP-3 mRNA following TGF- treatment thus appears to account for the increase in IGFBP-3 levels in conditioned media. The picture that emerges from these data and other studies is as follows; TGF- induces increases in IGFBP-3 mRNA (less than 8 h) and secreted IGFBP-3 levels (less than 2 days) as early events, followed by inhibition of monolayer cell growth (after 4 days) as a late event. These data are consistent with the hypothesis that TGF-2 inhibits proliferation, at least in part, by increasing IGFBP-3 expression.

To more directly test whether IGFBP-3 mediates TGF- action, we employed two different strategies to block the IGFBP-3 effect: 1) blocking TGF-2-induced IGFBP-3 expression by IGFBP-3 antisense ODN and 2) preventing IGFBP-3 binding to the cell surface by treatment with IGFs and IGF-II analogs, thereby blocking IGFBP-3-mediated action (4) . Cells were treated with antisense and sense phosphorothioate ODN corresponding to nucleotide positions 2021-2040 of the human IGFBP-3 cDNA sequence. Treatment with 10 µg/ml antisense, but not sense, ODN reduced IGFBP-3 protein concentrations by 80% and IGFBP-3 mRNA levels by 60% (Fig. 3A), but had no effect on IGFBP-4 production (data not shown). We next investigated the effects of antisense ODN on TGF-2-induced IGFBP-3 production. As in previous experiments, TGF-2 alone increased IGFBP-3 levels by as much as 4-fold. In the presence of 20 µg/ml antisense ODN, however, TGF-2-induced IGFBP-3 production was inhibited by as much as 80%, and this was correlated with 70% inhibition of TGF-2-induced IGFBP-3 mRNA expression at an ODN concentration of 20 µg/ml (Fig. 3B).


Figure 3: Effect of IGFBP-3 antisense ODN on IGFBP-3 protein and mRNA levels in control and TGF-2-treated cells. Upperpanel, Western ligand blots of Hs578T CM from cells treated with 1-20 µg/ml ODN on day 0 and again on day 1.5 without changing media in the absence (A) or presence (B) of 5 ng/ml TGF-2. CM was harvested after 3 days of treatment from triplicate wells within each experiment, pooled, and electrophoresed. Lowerpanel, Northern blots demonstrating the effects of ODN on IGFBP-3 mRNA levels in control (A) or TGF-2-treated (B) Hs578T cells. Cells were grown until 90% confluent and pretreated with 1-20 µg/ml ODN for 4 h. Cells were then washed and treated with ODN in the absence (A) or presence (B) of 5 ng/ml TGF-2 for 12 h. A representative gel from one of two experiments is shown.



After initially ascertaining the ability of the IGFBP-3 antisense ODN to specifically suppress TGF-2-induced IGFBP-3 production, we next investigated the effect of antisense ODN on TGF-2-induced growth inhibition by treating cells with antisense or sense ODN for 5 days. No significant growth effect was observed by IGFBP-3 antisense ODN in the absence of TGF-2 despite inhibition of IGFBP-3 expression, indicating that basal IGFBP-3 levels were not sufficient to exert a growth-inhibitory effect (data not shown). These findings are similar to our previous report that shown that IGFs had no effect on cell growth, despite their ability to block cell surface binding of endogenous IGFBP-3 (4) . However, when antisense ODN were employed in the presence of TGF-2, antisense, but not sense, ODN attenuated the TGF-2-induced cell growth inhibition by approximately 60% (p < 0.05) (Fig. 4).


Figure 4: Effect of antisense IGFBP-3 ODN on TGF-2-induced growth inhibition. Cells were treated with 5 ng/ml TGF-2 in the presence of antisense or sense ODN at concentrations ranging from 1 to 50 µg/ml. The inhibitory effect of TGF-2 was determined by cell counts after 5 days of treatment. Results represent means ± S.E. of two separate experiments performed in triplicate. *, p < 0.05.



We have previously identified IGFBP-3 receptors on Hs578T cells and demonstrated that blocking the interaction of exogenously added IGFBP-3 with these receptors by IGF peptides resulted in attenuation of IGFBP-3 inhibition of Hs578T cell growth (4) . Therefore, we tested the effect of IGF-II and IGF-II analogs on TGF-2 action (Fig. 5). Whereas TGF-2 alone resulted in a 40% inhibition of cell growth, when coincubated with IGF-II, the inhibitory effect was blocked in a dose-dependent manner, with a 90% attenuation of the TGF-2 effect at an IGF-II concentration of 200 ng/ml (p < 0.05). Similar results were observed using [Leu]IGF-II, which has full affinity for IGFBPs, but significantly reduced affinity for the IGF-I receptor (16) . In contrast, coincubation with similar concentrations of [Gln,Ala,Tyr,Leu,Leu]IGF-II ([QAYLL]IGF-II), an IGF analog with 100-fold reduced affinity for both IGFBPs and the IGF-I receptor, did not result in attenuation of the TGF-2 inhibitory effect. These results suggest that the attenuating effect of IGF-II and [Leu]IGF-II on TGF-2 inhibition of cell growth was not mediated through the IGF-I receptor, but instead reflects the ability of IGF peptides to prevent IGFBP-3 binding to the cell surface (4) .


Figure 5: Effect of IGF-II and IGF-II analogs on TGF-2 action. Cells were treated with IGF-II, [Leu]IGF-II, or [QAYLL]IGF-II at concentrations ranging from 5 to 200 ng/ml for 5 days. Results represent means ± S.E. of three separate experiments performed in triplicate. *, p < 0.05.




DISCUSSION

Previous studies have demonstrated that TGF- is a potent anti-proliferative factor in a series of human breast cancer cells in vitro(7, 21, 22) . Estrogen receptor (ER)-negative cells in particular, including Hs578T cells, appear to possess receptors specific for TGF-, and TGF-1 and TGF-2 appear to be equipotent in inhibiting cell growth (21, 22) . In addition, TGF- is produced and hormonally regulated in some ER-positive cells; anti-estrogens such as 4-OH-tamoxifen induce biologically active TGF- in MCF-7 cells, and this anti-estrogen-induced TGF- inhibits the growth of ER-negative cells (7) . Furthermore, a recent report has demonstrated that anti-estrogens, including 4-OH-tamoxifen, induce IGFBP-3 expression in MCF-7 cells (13) . The exact mechanism by which TGF- inhibits breast cancer cell proliferation is not well understood, despite the fact that TGF- receptors, at least type I and II receptors, are known to exhibit serine/threonine kinase activity (23) . It has been also demonstrated that stromal fibroblasts express TGF-, regardless of whether the fibroblasts originated from normal or malignant cells, indicating that TGF- is a tightly regulated negative growth factor in vivo(15) .

We have reported previously that exogenously added IGFBP-3 acts as a cell growth inhibitor, and that its inhibitory action may be mediated through specific IGFBP-3 receptors (4, 6) . In the present study, we demonstrate that physiological concentrations of TGF- specifically stimulate the expression of IGFBP-3 mRNA and protein. Furthermore, increased expression and production of IGFBP-3 precede the inhibition of cell growth by TGF-2 in Hs578T breast cancer cells. This result suggests that stimulation of IGFBP-3 expression is one of the earliest effects of the TGF- and, therefore, may be necessary for TGF--induced cell growth inhibition. Indeed, TGF--induced growth inhibition can be prevented by addition of IGFBP-3 antisense ODN or IGF peptides, but not by addition of sense ODN or IGF-II analogs that have reduced affinity for IGFBP-3.

In addition, recent studies have indicated that retinoic acid and the anti-estrogen tamoxifen stimulate production of IGFBP-3 and inhibit monolayer cell growth in human breast cancer cells (8, 13, 24, 25) , suggesting that IGFBP-3 may have a role in the growth-inhibitory action mediated by those factors in human breast cancer. Our IGFBP-3 antisense ODN and IGF-II analog data clearly demonstrate that TGF--induced growth inhibition is mediated, at least in part, through IGFBP-3 action. To the best of our knowledge, these studies constitute the first demonstration that IGFBP-3 is required for a growth-inhibitory effect that is induced by a different class of growth factors. A fuller elucidation of the mechanisms underlying these IGF-independent actions of IGFBP-3 in human breast cancer will allow us to understand how the growth of breast cancer cells can be modulated by the IGF/IGFBP system, and how other growth factors can interact with this system.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant CA58110 (to R. G. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Dept. of Pediatrics, L343, School of Medicine, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201-3042. Tel.: 503-494-1930; Fax: 503-494-1933.

Present address: Universitätskinderklink, D-97080 Würzburg, Federal Republic of Germany.

The abbreviations used are: IGFBP, insulin-like growth factor-binding protein; IGF, insulin-like growth factor; ER, estrogen receptor; TGF, transforming growth factor; ODN, oligodeoxynucleotide; DMEM, Dulbecco's modified Eagle's medium; CM, conditioned medium.


ACKNOWLEDGEMENTS

We thank C. Roberts (Oregon Health Sciences University) and P. Cohen (University of Pennsylvania, Philadelphia, PA) for critical reviews of the manuscript, Z. Gucev for technical assistance, and A. Sommer (Celtrix Inc., Santa Clara, CA) for providing IGFBP-3 and TGF-2.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.