Insulin-Like Growth Factor-I:Vitronectin Complex-Induced Changes in Gene Expression Effect Breast Cell Survival and Migration

Abstract

Recent studies have demonstrated that IGF-I associates with vitronectin (VN) through IGF-binding proteins (IGFBP), which in turn modulate IGF-stimulated biological functions such as cell proliferation, attachment, and migration. Because IGFs play important roles in transformation and progression of breast tumors, we aimed to describe the effects of IGF-I:IGFBP:VN complexes on breast cell function and to dissect mechanisms underlying these responses. In this study we demonstrate that substrate-bound IGF-I:IGFBP:VN complexes are potent stimulators of MCF-7 breast cell survival, which is mediated by a transient activation of ERK/MAPK and sustained activation of phosphoinositide 3-kinase/AKT pathways. Furthermore, use of pharmacological inhibitors of the MAPK and phosphoinositide 3-kinase pathways confirms that both pathways are involved in IGF-I:IGFBP:VN complex-mediated increased cell survival. Microarray analysis of cells stimulated to migrate in response to IGF-I:IGFBP:VN complexes identified differential expression of genes with previously reported roles in migration, invasion, and survival (Ephrin-B2, Sharp-2, Tissue-factor, Stratifin, PAI-1, IRS-1). These changes were not detected when the IGF-I analogue ([L(24)][A(31)]-IGF-I), which fails to bind to the IGF-I receptor, was substituted; confirming the IGF-I-dependent differential expression of genes associated with enhanced cell migration. Taken together, these studies have established that IGF-I:IGFBP:VN complexes enhance breast cell migration and survival, processes central to facilitating metastasis. This study highlights the interdependence of extracellular matrix and growth factor interactions in biological functions critical for metastasis and identifies potential novel therapeutic targets directed at preventing breast cancer progression.

Antagonists of IGF:Vitronectin Interactions Inhibit IGF-I–Induced Breast Cancer Cell Functions

“…The previously used substrate-bound approach (3,5,22) was adopted to investigate the effects of L 27 -IGF-II and candidate peptides on: the formation of IGF-I:IGFBP:VN complexes; complex-induced cell signaling; as well as monolayer cell viability and Transwell migration assays. IGF-I, IGFBP-3 and VN mixed 1:1:1 at 10 nmol/L each (IGF-I:IGFBP-3:VN complex), with or without the indicated concentrations of L 27 -IGF-II or peptides, were added to the culture-ware and incubated for 3 hours to allow complex formation.…”

“…Migration was assessed as described previously (5,22). Briefly, subconfluent cells were serum-starved for 4 hours and 6 Â 10 4 cells suspended in serum-free medium (SFM) were seeded onto the upper-surface of 8-mm-pore Transwell inserts (Corning Inc.) precoated on the under-surface with VN and IGF-I:IGFBP-3:VN, either alone or in combination with L 27 -IGF-II/peptides.…”

“…Cell viability was assessed as described before (3,5). Briefly, log phase cells were serum-starved for 4 hours and 5 Â 10 3 cells suspended in SFM were seeded into 96-well plates precoated with VN and IGF-I:IGFBP-3:VN, either alone or in combination with L 27 -IGF-II/peptides.…”

“…3,5,22), resolved on NuPAGE 4% to 12% gradient SDS-PAGE gels (Life Technologies) and transferred onto nitrocellulose membranes (Pall Corporation). The membranes were probed with anti-phospho ERK1/2 (1:1,000), anti-phospho AKT (1:5,000) and anti-phospho IGF-1Rb (1:1,000) antibodies.…”

“…3,4) to stimulate cell migration and proliferation and promote the transition of cancer cells to a therapy-resistant and metastatic phenotype (5).…”