Adhesion and migration of extracellular matrix-stimulated breast cancer
Introduction
Cancer metastasis consists of a complex cascade of events, which ultimately allow for tumor cell escape and seeding of ectopic environments. Two important steps in this cascade involve the ability of cancer cells to adhere to extracellular matrix (ECM) components and subsequently migrate through them. Integrins are a large family of adhesion molecules which are primarily responsible for attachment to ECM components. Integrin receptors are transmembrane glycoproteins composed of heterodimeric alpha (α) and beta (β) subunits. There are at least 16 α and 8 β subunits forming more than 20 functional receptors [1]. The subunits determine not only the ligand specificity but also the type of signal transduction system that will be activated [2].
Expression of various integrin receptors on breast cancer cells is thought to be associated with differences in metastatic behavior [3]. Zutter et al. [4] found that a decreased expression of the α2β1 integrin dimer was associated with a more malignant breast cancer phenotype. Friedrichs et al. [5] observed that high levels of α6 integrin expression were identified in over 50% of the breast cancers studied and inversely correlated with patient survival. Mukhopadhyay et al. [6] observed that the expression of the α6 integrin subunit correlated with the metastatic potential in a number of different tumor cell lines.
It has been shown that the interaction of the ECM protein fibronectin with integrins initiates a cascade of reactions that up-regulate more than 30 genes likely to be important in morphogenesis and tumor progression 7, 8, 9. A large number of fibronectin receptors exist on different tumor and nontumor cells, including α3β1, α4β1, α5β1, α8β1, αvβ1, αvβ3, αIIbβ3, αvβ6, α4β7, and α?β8 [10]. Fibronectin has been shown to mediate both invasion and metastasis in murine melanoma cells [11] as well as in vitro invasion of glioma cells [12].
Tumor cell integrin binding to the ECM protein vitronectin has also been shown to play a role in tumor metastasis. The integrin receptors for vitronectin include αvβ1, αvβ3, αvβ5, and αIIbβ3 [13]. The vitronectin receptor αvβ3 has been shown to directly influence the migration of melanoma as well as the tumor’s progression 14, 15.
In the current study, integrin receptor-blocking antibodies and specific peptidomimetic inhibitor compounds were used to evaluate the relationship between integrin receptors and the adhesion and migratory properties conferred by fibronectin and vitronectin in two breast tumor cell lines. In addition, the fibronectin fragments FN 120, CS-1, and RGDC, previously shown to bind different integrin receptors and inhibit binding to fibronectin, were used to further evaluate specific integrin involvement. The cell lines studied have previously been shown to have a high rate of invasion through Matrigel and to be highly metastatic in nude mice models 16, 17. Previous investigators have also shown these cell lines to express the fibronectin and vitronectin binding integrins αv, β1, β5, β3, and αvβ5 at varying levels 18, 19.
In this study it was demonstrated that both the β1 integrin subunit and the αvβ3 or αvβ5 integrin dimer are involved in breast cancer cell adhesion and migration to fibronectin and vitronectin. Furthermore, no single inhibitory agent alone was able to reduce the adhesion and migration to fibronectin and vitronectin.
Section snippets
Materials
MDA-MB-231 and MDA-MB-435 breast tumor cell lines were obtained from the Lombardi Cancer Institute, courtesy of Shared Tissue Bank. Costar (Corning, Inc., Acton, MA) high binding 96-multiwell plates (#3590) were used to bind either vitronectin or fibronectin. Falcon migration chambers, 8 μm pore size, were purchased from Fisher Scientific (Pittsburgh, PA). McCoy’s 5A, fetal bovine serum (FBS), penicillin-streptomycin, gentamicin, sodium pyruvate, MEM amino acid (50X), MEM amino acid (100X), MEM
Adhesion of MDA-MB-231 and MDA-MB-435 cells to vitronectin
In this and subsequent experiments, multiwell plates were coated with the appropriate extracellular matrix component as described in the Methods section. The effect of increasing concentrations of immobilized vitronectin on MDA-MB-231 and MDA-MB-435 is shown in Fig. 1. Tumor cell binding increased with increasing vitronectin concentrations. The maximum A630 of MDA-MB-435 was nearly twice that of MDA-MB-231. The half-binding points for both MDA-MB-435 and MDA-MB-231 cells to vitronectin
Discussion
This investigation attempted to address the relationship between agents that block tumor cell adhesion to extracellular matrix (ECM) components such as vitronectin and fibronectin and the migratory properties of breast tumor cells. As summarized in Table 1, the anti-α5 (P1D6) and the anti-β1 (Mab-13) antibodies could reduce the fibronectin-induced migratory properties of MDA-MB-231 breast tumor cells (column 4) in addition to reducing the adhesion properties of that cell line (column 2).
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