Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-I-dependent signaling

Background: Lysophosphatidic acid (LPA) and sphingosine I-phosphate (SIP) are bioactive lipid signaling molecules implicated in tumor dissemination. Membrane-type matrix metalloproteinase 1 (MTI-MMP) is a membrane-tethered collagenase thought to be involved in tumor invasion via extracellular matrix degradation. In this study, we investigated the molecular requirements for LPA- and SIP-regulated tumor cell migration in two dimensions (2D) and invasion of three-dimensional (3D) collagen matrices and, in particular, evaluated the role of MT1-MMP in this process. Results: LPA stimulated while SIP inhibited migration of most tumor lines in Boyden chamber assays. Conversely, HT1080 fibrosarcoma cells migrated in response to both lipids. HT1080 cells also markedly invaded 3D collagen matrices (similar to 700 mu m over 48 hours) in response to either lipid. siRNA targeting of LPA(I) and RacI, or SIPI, RacI, and Cdc42 specifically inhibited LPA- or SIP-induced HT1080 invasion, respectively. Analysis of LPA- induced HT1080 motility on 2D substrates vs. 3D matrices revealed that synthetic MMP inhibitors markedly reduced the distance (similar to 125 mu m vs. similar to 45 mu m) and velocity of invasion (similar to 0.09 mu m/min vs. similar to 0.03 mu m/min) only when cells navigated 3D matrices signifying a role for MMPs exclusively in invasion. Additionally, tissue inhibitors of metalloproteinases (TIMPs)-2, -3, and -4, but not TIMP-1, blocked lipid agonist-induced invasion indicating a role for membrane-type (MT)-MMPs. Furthermore, MTI-MMP expression in several tumor lines directly correlated with LPA- induced invasion. HEK293s, which neither express MTI-MMP nor invade in the presence of LPA, were transfected with MTI-MMP cDNA, and subsequently invaded in response to LPA. When HT1080 cells were seeded on top of or within collagen matrices, siRNA targeting of MTI-MMP, but not other MMPs, inhibited lipid agonist-induced invasion establishing a requisite role for MTI-MMP in this process. Conclusion: LPA is a fundamental regulator of MTI-MMP-dependent tumor cell invasion of 3D collagen matrices. In contrast, SIP appears to act as an inhibitory stimulus in most cases, while stimulating only select tumor lines. MTI-MMP is required only when tumor cells navigate 3D barriers and not when cells migrate on 2D substrata. We demonstrate that tumor cells require coordinate regulation of LPA/SIP receptors and Rho GTPases to migrate, and additionally, require MTI-MMP in order to invade collagen matrices during neoplastic progression.