Insulin-Like Growth Factor Binding Protein 3 Has Opposing Actions on Malignant and Nonmalignant Breast Epithelial Cells that Are Each Reversible and Dependent upon Cholesterol-Stabilized Integrin Receptor Complexes

Abstract

IGF-binding protein (IGFBP)-3 is generally considered to have actions that counterbalance those of IGFs and is therefore being developed as a cancer treatment. In breast tumors, however, high levels are associated with aggressive tumors and poor prognosis. Consistent with this we have demonstrated that although IGFBP-3 and a non-IGF-binding fragment (serine phosphorylation domain peptide) reduced attachment and enhanced apoptosis of Hs578T breast cancer cells cultured on collagen or laminin, it promoted their attachment and survival on fibronectin, which is abundant in the matrix of aggressive tumors. We have now examined the factors that determine whether IGFBP-3 has positive or negative actions on breast epithelial cells. IGFBP-3 also promoted survival of Hs578T cells in the presence of an antibody to the beta1-integrin subunit or when cholesterol-stabilized complexes were disrupted. These actions were blocked by IGF-I or a MAPK inhibitor. Serine phosphorylation domain peptide had similar actions on MCF-7 cells that were again reversed on fibronectin or with disruption of cholesterol-stabilized complexes and blocked by the beta1-integrin antibody. In contrast, IGFBP-3 promoted growth and survival for nonmalignant MCF-10A cells, but these effects were again reversed on fibronectin and blocked by the beta1 antibody or a MAPK inhibitor or by disruption of cholesterol-stabilized complexes. On Hs578T cells, IGFBP-3 bound to caveolin-1 and beta1-integrins, enhancing their aggregation, the recruitment of focal adhesion kinase, and the activation of MAPK. In summary, with three breast epithelial cell lines, IGFBP-3 had positive or negative effects on growth and survival dependent upon the status of cholesterol-stabilized integrin receptor complexes.

IGFBP-3 Can Either Inhibit or Enhance EGF-mediated Growth of Breast Epithelial Cells Dependent upon the Presence of Fibronectin

“…These interactions are very analogous to those that we have found between IGFBP-3 and EGF. It has been reported that EGF regulates IGFBP-3 production (21, 22), and we have described in this study and previously that that the intrinsic effects of IGFBP-3 on cell growth also involves integrin receptor activation and phosphorylation of MAPK culminating in cell proliferation (5).…”

“…In addition there have been several reports that in breast tumors the expression of IGFBP-3 is positively associated with large, highly proliferative tumors and poor prognostic markers (14,15). Furthermore, we have previously reported that in contrast to its inhibitory effects on breast cancer cells, IGFBP-3 promoted the proliferation and survival of the relatively normal, non-malignant, anchoragedependent MCF-10A cells (12), which we also showed was dependent upon ␤1 integrins and subsequent activation of MAPK (5).…”

“…Cholesterol-stabilized complexes are required for normal integrin signaling, and we showed that disrupting such complexes also reversed the intrinsic action of IGFBP-3 (5). These reports challenge the widely held view that IGFBP-3 normally has inhibitory actions and suggest that its actions may depend not just on cell type but also on cell context.…”

“…No specific cell surface receptors have been described for CCN proteins, but it is now recognized that they generally act via integrin receptors with which they interact through non-classical recognition sequences (56). Although IGFBP-3 also does not possess a classical integrin recognition sequence, it has been reported to associate with the ␤1 integrin (5,49) and to bind with high affinity to many other proteins that are recognized to interact directly with integrins, including fibronectin (57), fibrin and fibrinogen (58), plasminogen (59), ADAM12 (60), caveolin-1, and the transferrin receptor (49,61). In support of this, a recent report has demonstrated that IGFBP-3 potentiation of EGF-induced growth in the nonmalignant MCF10A cells involves activation of sphingosine kinase 2, which resulted in sphingosine-1 phosphate production acting on sphingosine 1-phosphate 1/3 receptors (62).…”

“…4). We then defined a pathway by which IGFBP-3 could elicit these effects on apoptosis of breast epithelial cells: IGFBP-3 bound to ␤1 integrins and caveolin 1 and increased their association, which culminated in the recruitment of focal adhesion kinase and the subsequent activation of MAPK (5).…”