Insulin-Like Growth Factor-II Bound to Vitronectin Enhances MCF-7 Breast Cancer Cell Migration

0013-7227/03/$15.00/0 Printed in U.S.A. Endocrinology 144(6):2417–2424 Copyright © 2003 by The Endocrine Society doi: 10.1210/en.2002-221138 Insulin-Like Growth Factor-II Bound to Vitronectin Enhances MCF-7 Breast Cancer Cell Migration ANTHONY NOBLE, CHRIS TOWNE, LISA CHOPIN, DAVID LEAVESLEY, AND ZEE UPTON Tissue BioRegeneration and Integration Research Program, Center for Molecular Biotechnology, School of Life Sciences, Queensland University of Technology, Brisbane, Queensland 4000, Australia We have previously reported that IGF-II binds the extracellular matrix protein vitronectin (VN) with an affinity similar to that for the type-1 IGF receptor (IGF-1R). In view of this finding, and given the cited role of VN in cell motility and adhesion, we aimed to elucidate the functional consequences of this interaction on cellular processes relevant to breast carcinoma. We demonstrate that this complex slightly inhibits cell attachment and has little effect on protein synthesis in MCF-7 breast cancer cells. However, prebinding IGF-II to immobilized VN was found to significantly enhance breast cancer cell migration through Transwells. Interestingly, IGF-II I GF-I and IGF-II ARE mitogenic peptide growth factors involved in a broad range of cellular processes, including hyperplasia, DNA synthesis, differentiation, cell cycle progression, and inhibition of apoptosis (1–3). These effects are mediated through binding to their tyrosine-kinase-linked cell surface receptor, the type 1 IGF receptor (IGF-1R). The IGFs are also tightly regulated by a family of specific binding proteins, termed IGF-binding proteins (IGFBPs), whose primary role is to bind free IGFs and thereby moderate their half-life, specificity, and activity (4). The IGFs have previously been linked with processes related to breast cancer progression and metastasis, such as cell cycle control, inhibition of apoptosis, and cell migration (3). In addition, loss of control of the IGF system is believed to be highly relevant to cancer progression. For example, high levels of the IGF-I and IGF-1R and low IGFBP levels are associated with poor long-term prognosis in breast cancer (5, 6). Altered contact between cells and their associated extracellular matrix (ECM) is a prerequisite for breast cancer cell migration and, thus, metastasis (7). Cell migration requires interactions with adhesive molecules, such as vitronectin (VN). Indeed, interaction of cells with VN has been reported to be essential for IGF-stimulated migration (8 –11). Recently, VN has been shown to bind directly to IGF-II (12), whereas IGF-I can bind to VN in the presence of select IGFBPs (13, 14). The finding that VN, an ECM organization and adhesion molecule, binds IGF-II with an affinity that is similar to that of IGF-II for IGF-1R (12), its biologically relevant receptor, reveals a specific physical link between IGF action and VN Abbreviations: CIMPR, Cation-independent mannose-6-phohosphate receptor; DMEM/F12, DMEM/Ham’s F12; ECM, extracellular matrix; ER, estrogen receptor; HBB, HEPES binding buffer; IGFBP, IGFbinding protein; IGF-1R, type-1 IGF receptor; SFM, serum-free media; VN, vitronectin. bound to VN, and not IGF-II in solution in the presence of VN, seems to be responsible for the effects on cell migration. Furthermore, studies using analogs of IGF-II with reduced affinity for the IGF-1R or IGF binding proteins indicate that this response involves the IGF-1R but is independent of IGF binding proteins. This is the first study demonstrating that IGFII:VN complexes enhance migration of cells. This may prove to be especially relevant, given that overexpression of IGF-II and VN are features of many tumors. (Endocrinology 144: 2417–2424, 2003) in the ECM. In addition, recent studies in our laboratory have demonstrated that IGF-II bound to VN can stimulate synergistic functional responses in human keratinocytes in vitro (13). Taken together, these findings suggest that the formation of IGF-II:VN complexes may play an important role in modulating the effect of IGF-II in VN-rich ECM. VN is a glycoprotein that is highly abundant in the blood and in the ECM. Primarily synthesized in the liver, but expressed by many other cell types, VN circulates in the blood in a closed conformation and is deposited in the ECM in an open, or extended, conformation (15). Both conformations are believed to bind IGF-II (12, 13, 16) and also bind multiple other ligands, including collagen (17), glycosaminoglycans (18), many other ECM proteins, and a wide variety of integrins, particularly the ␣v-integrins. Indeed, the primary role of VN is as an ECM organization molecule that provides adhesive links to these cell-surface integrin receptors via an RGD-binding motif. The VN receptors (␣v-integrins) have been shown to regulate the actin cytoskeleton rearrangement required for growth and invasion; hence, VN binding coordinates cell adhesion and movement (19, 20). VN has been implicated in a number of cellular processes associated with cancer. These range from control of metastasis (21) to reattachment at remote sites (15) and angiogenesis (22). Moreover, metastatic cancer cells have increased expression of ␣v-integrins, and an increased expression of VN correlates with metastatic potential in gliomas and colorectal adenocarcinomas (23, 24). Hence, loss of controlled adhesion, by up-regulation of VN and its receptors, may be a key determinant of metastatic ability (25, 26). IGF-II has been demonstrated to stimulate increased migration in breast carcinoma cells (9). One mechanism through which this has been hypothesized to occur is by increased ␣v␤3-expression, thus facilitating an increased interaction with VN in the ECM. Studies examining functional interac- 2417 2418 Endocrinology, June 2003, 144(6):2417–2424 tions between VN and the IGF system demonstrate that VN and IGF are tightly regulated and closely associated. This is illustrated by demonstration that occupancy of both the ␣v␤3-VN receptor and the IGF-1R are prerequisites for breast cancer cell migration (9, 10, 27) and by the finding that the insulin receptor substrate 1, a downstream target of IGF-1R, associates with the VN receptors, the ␣v-integrins (28). Furthermore, breast tissues are known to be highly responsive to IGFs and are exposed to extensive hormone-stimulated matrix remodeling in their normal life cycle. Changes in these systems are also relevant in the cancer phenotype. The high level of interaction between VN and the IGF system in breast cancer, along with the demonstrated synergy of IGFs complexed to VN in stimulating keratinocyte protein synthesis (13), suggests that the IGF-II:VN complex will be biologically relevant in breast cancer cells, perhaps modulating tumor development and progression. This study aimed to examine the effect of the IGF-II:VN complex on processes important in breast cancer development and progression. Thus, the attachment, protein synthesis, and migration of poorly me (...truncated)