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

    Copyright 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain.

    0955-2235/95 $29.00 + .00




    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 of 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-inde- pendent 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 of 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 oligodeoxynu- cleotide not only blocks TGF-fl and RA simulation of 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.


  • 504 Youngman Oh et al.


    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 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 expres- sion and ligand binding assays, have demonstrated that virtually all the specimens examined express and produce all three receptors [17-20]. Using estrogen-depen- dent 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 mecha- nisms 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 secre- tion 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 recep- tors. 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 sig- nificance and the mechanisms involved in IGFBP-3 proteolysis in human breast cancer cells are unclear.


    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.cIi 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 enzymati- cally removed by pretreatment with heparinase or heparitinase for 5 h at 37C, 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.

  • 506 Youngman Oh et al.

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