Download - Antiproliferative actions of insulin-like growth factor binding protein (IGFBP)-3 in human breast cancer cells

~ ) Pergamon Progress in Growth Factor Research, Vol. 6. Nos. 2-4, pp. 503-512, 1995

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ANTIPROLIFERATIVE ACTIONS OF INSULIN-LIKE GROWTH FACTOR BINDING PROTEIN (IGFBP)-3

IN HUMAN BREAST CANCER CELLS

Youngman Oh,* Zoran Gucev, Lilly Ng, Hermann L. Mi~ller and Ron G. Rosenfeld

Department of Pediatrics, School of Medicine, Oregon Health Sciences University, Portland, OR 97201-3042, U.S.A.

A number of lines of evidence suggest that IGFs are important mitogens in human breast cancer: (1) IGFs are the most potent growth factor in human breast cancer cells; (2) estrogen stimulates expression of IGF-H and the type 1 IGF receptor; and (3) stromal cells express IGFs, which may act in a paracrine manner. Numerous studies have demonstrated that IGFBPs modulate the mitogenic effects o f IGFs in the local environment. In particular, we have recently demonstrated that IGFBP-3 inhibits the growth of Hs578T and MDA-MB-231 human breast cancer cells in an IGF-independent manner. Further studies revealed the existence of cell surface-associated IGFBP-3 receptors. Receptor binding and the subsequent antiproliferative action of IGFBP-3 was inhibited by IGFs, owing to the formation o f an IGF-IGFBP-3 complex that prevents the binding of lGFBP-3 to its receptors. In addition, exogeneously added soluble heparin or heparan sulfate inhibited the binding of IGFBP-3 to the cell surface in a dose-dependent manner. However, when heparin and heparan sulfate linkages of glycosaminoglycans on the cell surface were enzymatically removed, IGFBP-3 binding was only minimally affected. These data suggest that soluble heparin or heparan sulfate forms a complex with IGFBP-3, thereby inhibiting receptor binding of IGFBP-3, rather than competing with cell-surface glycosaminoglycans for binding of 1GFBP-3.

Additionally, the role of IGFBP-3 in the antiproliferative effects of transforming growth factor (TGF)-~ and retinoic acid (RA) is supported by our observations that: (1) inhibition of lGFBP-3 gene expression using an IGFBP-3 antisense oligodeoxynucleotide not only blocks TGF-fl and RA simulation o f IGFBP-3 production by up to 90%, but also inhibits their antiproliferative effects by 40--60%; and (2) treatment with IGF-H and IGF-H analogs diminish TGF-~ effects by blocking TGF-fl induced binding of IGFBP-3 to the cell surface.

Taken together, our results support the hypothesis that IGFBP-3 is an important antiproliferative factor in human breast cancer, acting in an IGF-independent manner in addition to its ability to modulate the binding of lGF peptides to IGF receptors.

Keywords: IGFBP-3, breast cancer, antiproliferation, IGFBP-3 receptor, glycosaminoglycan,

*Correspondence to: Youngman Oh, Ph.D., Tel: (503) 494-1930; Fax: (503) 494-1933.

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504 Youngman Oh et al.

INTRODUCTION

Breast tumors express variable levels of receptors for steroids [1-3] and growth factors [4--6] and it has been proposed that the ligands for these receptors act through a variety of autocrine and paracrine mechanisms. Studies in vivo and in cell culture systems have implicated a number of steroids and polypeptide growth factors in the regulation of human breast cell replication [7-10] but the relative contribution of each growth factor and the precise mechanism by which each acts are not known. At the present time, several growth factors have also been identified as playing a major role in the regulation of mammary growth, including members of the epidermal growth factor/transforming growth factor a (EGF/TGF-a) family, IGFs, heparin-binding growth factors (fibroblast growth factors (FGFs) and others), and the TGF-/3 family.

THE IGF AXIS IN THE HUMAN MAMMARY SYSTEM

The IGFs have been recognized as major regulators of mammary epithelial cell and breast cancer cell growth [5, 6, 11, 12]. Nevertheless, limited information is currently available on the IGF axis (IGFs, IGFBPs and IGF receptors) in breast cancer. Both IGF-I and IGF-II have been shown to be potent mitogens for a number of breast cancer cell lines in vitro [5, 6, 12-14], and IGF-I and IGF-II, mRNA is detectable in normal and malignant human mammary cells [15, 16]. In addition, studies of the type 1 and type 2 IGF receptors and insulin receptors in breast tumor specimens and breast cancer cell lines, employing both mRNA expression and ligand binding assays, have demonstrated that virtually all the specimens examined express and produce all three receptors [17-20]. Using estrogen-dependent breast cancer cells, it has been further demonstrated that the mitogenic effects of both IGF-I and IGF-II are mediated by the type 1 IGF receptor [5, 6].

Breast cancer cells also secrete various types of IGFBPs. The molecular mechanisms and biological functions involved in the interaction of the IGFBPs with the IGFs remain unclear, but these molecules appear to regulate the availability of free IGFs for interaction with IGF receptors [21]. The predominant secreted IGFBP appears to correlate with the estrogen receptor status of the cell [22, 23]. Estrogen- non-responsive (ER-negative) cells predominantly secrete IGFBP-3 and IGFBP-4 as major species, and IGFBP-6 as a minor binding protein, whereas estrogen- responsive (ER-positive) cells secrete IGFBP-2 and IGFBP-4 as major species, and IGFBP-3 and IGFBP-5 as minor proteins. These different patterns of IGFBP secretion in two different classes of breast cancer cells imply that the IGF system in breast cancer is complex, and that the biological significance and determination of the cellular response of IGFBPs to autocrine, paracrine, or endocrine-derived IGFs may be significantly different, depending on estrogen responsiveness.

IGFBP-3 is the principal IGFBP in adult serum, where it circulates as a 150 kDa complex consisting of IGFBP-3, an acid-labile subunit, and IGF peptide [24, 25]. Its principal role has been postulated to be the transport of IGFs, protecting them from rapid clearance and/or degradation [26, 27]. In human breast cancer cells, expression of IGFBP-3 is hormonally regulated: (1) estrogen inhibits expression of IGFBP-3 in ER-positive cells, whereas antiestrogens stimulate production of

Inhibition of Breast Cancer Growth by IGFBP-3 505

IGFBP-3 [28]; and (2) TGF-fl and RA stimulate IGFBP-3 production [29, 30]. Furthermore, post-translational modification of IGFBP-3 has been observed; specifically, IGFBP-3 can be proteolyzed by proteases such as cathepsin D, prostate-specific antigen (PSA) and plasmin, all of which have been detected in human breast cancer cells [31-36]. In general, IGFBP-3 proteases are postulated to play a role in altering tissue IGF availability by lowering the affinity of IGFBP-3 for its ligand, thereby increasing the availability of IGFs to cell-membrane receptors. PSA, for example, has been shown to reverse the inhibitory effect of IGFBP- 3 on stimulated prostate cell growth by cleaving IGFBP-3 and generating IGFBP-3 fragments that exhibit a lower affinity for IGFs [34]. However, the biological significance and the mechanisms involved in IGFBP-3 proteolysis in human breast cancer cells are unclear.

IGF-INDEPENDENT ACTION OF IGFBP-3 IN THE HUMAN BREAST CANCER CELLS

Series of our previous studies have demonstrated the presence of cell surface- associated IGFBP-3 on Hs578T ER-negative human breast cancer cells [37], and that exogeneous IGFBP-3 specifically binds to the cell surface and inhibits cell monolayer growth, by itself, through an IGF-independent mechanism [38]. Binding of [125I]-IGFBP-3E- cot~ was specific and could not be displaced by unlabeled IGFBP- 1 or fibronectin. As IGFBP-3 possesses a putative 'heparin-binding motif' near its C-terminus, we tested whether the binding of IGFBP-3 to the Hs578T cell surface resulted in interaction with heparin-containing glycosaminoglycans. Exogenously added soluble heparin and heparan sulfate inhibited [125I]-IGFBP-3E.c°Ii binding to the cell surface in a dose-dependent manner with half maximal inhibition at l0 and >_.250 pg m1-1 respectively, whereas chondroitin sulfate A showed no inhibitory effect at concentrations up to 250 /.tg m1-1 (Fig. 1). However, when heparin and heparan sulfate linkages of glycosaminoglycans on the cell surface were enzymatically removed by pretreatment with heparinase or heparitinase for 5 h at 37°C, IGFBP-3 binding was minimally affected (Fig. 1). These data suggest that soluble heparin or heparan sulfate forms a complex with IGFBP-3, thereby inhibiting binding of IGFBP-3 to cell-surface protein(s) specific to IGFBP-3, rather than competing with cell-surface glycosaminoglycans for binding of IGFBP-3.

These findings suggested the existence of specific cell-surface association proteins or receptors for IGFBP-3 on Hs578T human breast cancer cells. Furthermore, IGF-I and IGF-II can attenuate the inhibitory effect of IGFBP-3 by forming IGF- IGFBP-3 complexes, thereby preventing cell-surface binding of IGFBP-3 [38]. In addition, we have recently demonstrated the presence of putative IGFBP-3 specific receptors that are capable of mediating the direct inhibitory effect of IGBBP-3 on human breast cancer cell growth [39]. These 20, 26, and 50 kDa putative IGFBP-3 receptors have been further purified by IGFBP-3-anti-IGFBP-3 antibody immuno- affinity membranes.


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FIGURE 1. Effect of heparin and heparan sulfate on [I~I]-IGFBP-3 e'c°ti binding to Hs578T monolayers. Cells were grown to confluence 24-multiwell plates and maintained in serum-free media. Cells were incubated for 3 h at 15°C with 50,000 cpm [125I]-IGFBP-3e.c°n in the presence of various concentrations of heparin, heparan sulfate and chondroitin sulfate A. Ceils were then washed, and ceil-associated radio- activity determined. To enzymatically remove heparin and heparan sulfate linkages of glycosaminoglycaus on the cell surface, cells were pretreated with various concentrations of heparinase and heparitinase (data not shown) for 5 h, 27°C.

THE ROLE OF IGFBP-3 IN TGF-~- AND RA-INDUCED GROWTH INHIBITION IN BREAST CANCER CELLS

TGF-fl and RA have been identified as potent antiproliferative factors in human breast cancer cells in vitro. However, TGF-fl and RA have been found to stimulate IGFBP-3 production in different cell systems in which they inhibit proliferation, suggesting that IGFBP-3 may act as a mediator of their antiproliferative effects in human breast cancer cells. In estrogen receptor-negative human breast cancer cells (Hs578T and MDA-MB-231), several lines of evidence support this hypothesis. Our studies have demonstrated that TGF-fl and RA stimulate IGFBP-3 gene expression (2- fold) and production (2-3-fold) prior to their inhibition of cell growth in Hs578T and MDA-MB-231 cells (data not shown). To test more directly whether IGFBP-3 medi- ates TGF-fl and RA action, we employed two different strategies to block the IGFBP- 3 effect: (1) blocking TGF-fl induced IGFBP-3 expression with an IGFBP-3 antisense oligodeoxynucleotide (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 [40]. Cells were treated with antisense and sense ODN corresponding to nucleotide positions 2021 to 2040 of the human IGFBP-3 cDNA sequence. Treatment with 10/.tg ml -~ antisense, but not sense, ODN reduced basal IGFBP-3 protein concentrations by 80% and IGFBP-3 mRNA levels by 60% in Hs578T [40] and MDA-MB- 231 cells (data not shown). Further experiments revealed that inhibition of IGFBP-3 gene expression using an IGFBP-3 antisense O D N not only blocks TGF-fl and RA stimulation of IGFBP-3 production but also inhibits their antiproliferative effects. In Hs578T cells, TGF-fl2-induced IGFBP-3 production was inhibited by as much as 80%, and this was correlated with 70% inhibition of TGF-fl2-induced IGFBP-3 mRNA expression in the presence of 20 pg ml -~ antisense ODN (Fig. 2). Similar effects


IGFBP-3 protein

IGFBP-3 mRNA

actin

+ TGF- ~2 (ng/ml) 0 0 5 5 5 5 5 5 5 + Antisense (ng/ml) + Sense 0 0 0 1 10 20 1 10 20

FIGURE 2. Effect of IGFBP-3 antisense ODN on IGFBP-3 protein and mRNA levels in TGF-~2 - treated Hs578T cells. Upper panel: Western ligand blots of Hs578T CM from cells treated with 1 to 20 #g m1-10DN on day 0 and again on day 1.5 without changing media in the presence of 5 ng m1-1 of TGF-~2. CM was harvested after 3 days of treatment from triplicate wells within each experiment, pooled, and elec- trophoresed. Lower panel: Northern blots demonstrating the effects of ODN on IGFBP-3 mRNA levels in TGF-~2 treated Hs578T cells. Cells were grown until 90% confluent and pretreated with 1 to 20 Ilg ml -I ODN for 4 h. Cells were then washed and treated with ODN in the presence of 5 ng m1-1 of TGF-[12 for 12 h. A representative gel from one of two experiments is shown [40].

of the IGFBP-3 antisense ODN were observed in MDA-MB-231 cells; treatment with 20/zg ml -t antisense ODN resulted in >90% inhibition on TGF-/32-induced IGFBP-3 production and mRNA expression. In addition, RA-induced IGFBP-3 production and mRNA expression were inhibited by as much as 90% in the presence of 1 /zg ml -t IGFBP-3 antisense ODN in MDA-MB-231 cells (data not shown).

After initially ascertaining the ability of the IGFBP-3 antisense ODN to suppress TGF-fl2 and RA-induced IGFBP-3 production, we next investigated the effect of the IGFBP-3 antisense ODN on TGF-132- and RA-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-fl2 or RA despite inhibition of IGFBP-3 expression, indicating that basal IGFBP-3 levels were not sufficient to exert a growth-inhibitory effect in both cell lines (data not shown). However, when antisense, but not sense, ODN were employed in the presence of TGF-fl2 (Hs578T cells) or RA (MDA-MB-231 cells), antisense ODN attenuated the TGF-fl2- and RA-induced cell growth inhibition by approximately 60% and 40%, respectively (P<0.05) (Fig. 3).

As demonstrated previously, blocking the interaction of exogenously added IGFBP-3 with IGFBP-3 receptors by the addition of IGF peptides resulted in attenuation of IGFBP-3 inhibition of Hs578T cell growth. Similar effects of IGF peptides on TGF-fl induced cell growth inhibition were observed in Hs578T cells (Fig. 4). Whereas TGF-fl2 alone resulted in a 40% inhibition of cell growth, when coincubated with IGF-II, the inhibitory effect was blocked in a dose-dependent


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+ RA 10.5 pM) FIGURE 3. Effect of IGFBP-3 antisense ODN on TGF-~2- and RA-induced growth inhibition. Treatment of Hs578T cells with 5 ng m1-1 TGF-~2 (A) or MDA-MB-231 cells with 0.5 p M RA (B) in the presence of antisense or sense ODN at concentrations ranging from 1 to 50 pg ml -l. The inhibitory effect of TGF-~2 and RA 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.

manner, with a 90% attenuation of the TGF-fl2 effect at an IGF-II concentration of 200 ng ml -l (P<0.05). Similar results were observed using [LeuZ7]IGF-II which has full affinity for IGFBPs, but significantly reduced affinity for the IGF-I receptor [40]. In contrast, co-incubation with similar concentrations of [Gln 6, Ala 7, Tyr 18, Leu 19, Leu 27] IGF-II ([QAYL-L]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-fl2 inhibitory effect. These results suggest that the attenuating effect of


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IGF-II and [Leu27]IGF-II on TGF-fl2 inhibition of cell growth was not mediated through the IGF-I receptor, but rather, reflects the ability of IGF peptides to prevent IGFBP-3 binding to the cell surface.

Our studies indicate that endogenously produced IGFBP-3 is an important direct inhibitor of proliferation of human breast cancer cells, and importantly, that a major mechanism in the inhibitory actions of TGF-fl and RA on these cells appears to be their stimulation of cellular production of IGFBP-3, which then exerts direct antiproliferative effects via interaction with IGFBP-3 specific receptors.

In summary, we have: (1) demonstrated specific and high-affinity association of IGFBP-3 with human breast cancer cell surface; (2) demonstrated that binding of IGFBP-3 to breast cancer cells results in a major IGF-independent antiproliferative effect; (3) shown that transcriptional regulation of IGFBP-3 explains, at least in part, the antiproliferative effects of TGF-fl and RA in ER-negative human breast cancer cells.

Taken together with other studies on ER-positive breast cancer cells that demon- strate that the cell growth effects of estrogen and antiestrogens are also mediated, in part, through modulation of IGFBP-3 expression [42], we hypothesize that IGFBP-3 inhibits human breast cancer growth by acting through its specific receptor, in addition to regulating the binding of IGF peptides to classical IGF receptors in human breast cancer cells (Fig. 5). Furthermore, we believe that the IGFBP- 3/IGFBP-3 receptor system exists widely as a growth-suppressing factor in both normal and malignant breast cancer cells and that abnormalities of the IGFBP- 3/IGFBP-3 receptor axis may contribute to the etiology of breast cancer. Therefore, a fuller understanding of IGFBP-3 and the IGFBP-3-receptor interaction will allow us to design IGFBP-3 agonists that have potential therapeutic value. In particular,


IGFBP-3 Q Q Q (4) IGFBP'3 PrOt'as-

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FIGURE 5. Schematic diagram of mechanisms for inhibitory action of IGFBP-3. IGFBP-3 inhibits human breast cancer cell growth by: (1) acting through its own receptor; (2) blocking the binding of IGF peptides to the Type I IGF receptor; (3) acting as a major element for anti-proliferation growth factors, such as TGF-~, retinoic acid; (4) acting as a substrate for IGFBP-3 proteases; and (5) mediating antiestrogen-induced growth inhibition in ER-positive breast cancer cells.

t r e a t m e n t wi th I G F B P - 3 agon i s t s m a y be espec ia l ly v a l u a b l e in E R - n e g a t i v e o r

m u l t i - d r u g res i s tan t b reas t cancers .

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Download - Antiproliferative actions of insulin-like growth factor binding protein (IGFBP)-3 in human breast cancer cells

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