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Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by the membrane-anchored glycoprotein RECK.

ABSTRACT: A human fibroblast cDNA expression library was screened for cDNA clones giving rise to flat colonies when transfected into v-Ki-ras-transformed NIH 3T3 cells. One such gene, RECK, encodes a membrane-anchored glycoprotein of about 110 kDa with multiple epidermal growth factor-like repeats and serine-protease inhibitor-like domains. While RECK mRNA is expressed in various human tissues and untransformed cells, it is undetectable in tumor-derived cell lines and oncogenically transformed cells. Restored expression of RECK in malignant cells resulted in suppression of invasive activity with concomitant decrease in the secretion of matrix metalloproteinase-9 (MMP-9), a key enzyme involved in tumor invasion and metastasis. Moreover, purified RECK protein was found to bind to, and inhibit the proteolytic activity of, MMP-9. Thus, RECK may link oncogenic signals to tumor invasion and metastasis.

SUBMITTER: Takahashi C

PROVIDER: S-EPMC23764 | biostudies-literature | 1998 Oct

REPOSITORIES: biostudies-literature

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Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by the membrane-anchored glycoprotein RECK.

Takahashi C C Sheng Z Z Horan T P TP Kitayama H H Maki M M Hitomi K K Kitaura Y Y Takai S S Sasahara R M RM Horimoto A A Ikawa Y Y Ratzkin B J BJ Arakawa T T Noda M M

Proceedings of the National Academy of Sciences of the United States of America 19981001 22

A human fibroblast cDNA expression library was screened for cDNA clones giving rise to flat colonies when transfected into v-Ki-ras-transformed NIH 3T3 cells. One such gene, RECK, encodes a membrane-anchored glycoprotein of about 110 kDa with multiple epidermal growth factor-like repeats and serine-protease inhibitor-like domains. While RECK mRNA is expressed in various human tissues and untransformed cells, it is undetectable in tumor-derived cell lines and oncogenically transformed cells. Rest ...[more]

PMID: 9789069

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Project description:BackgroundLysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are bioactive lipid signaling molecules implicated in tumor dissemination. Membrane-type matrix metalloproteinase 1 (MT1-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 S1P-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.ResultsLPA stimulated while S1P 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 (approximatly 700 microm over 48 hours) in response to either lipid. siRNA targeting of LPA1 and Rac1, or S1P1, Rac1, and Cdc42 specifically inhibited LPA- or S1P-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 (approximately 125 microm vs. approximately 45 microm) and velocity of invasion (approximately 0.09 microm/min vs. approximately 0.03 microm/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, MT1-MMP expression in several tumor lines directly correlated with LPA-induced invasion. HEK293s, which neither express MT1-MMP nor invade in the presence of LPA, were transfected with MT1-MMP cDNA, and subsequently invaded in response to LPA. When HT1080 cells were seeded on top of or within collagen matrices, siRNA targeting of MT1-MMP, but not other MMPs, inhibited lipid agonist-induced invasion establishing a requisite role for MT1-MMP in this process.ConclusionLPA is a fundamental regulator of MT1-MMP-dependent tumor cell invasion of 3D collagen matrices. In contrast, S1P appears to act as an inhibitory stimulus in most cases, while stimulating only select tumor lines. MT1-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/S1P receptors and Rho GTPases to migrate, and additionally, require MT1-MMP in order to invade collagen matrices during neoplastic progression.

Project description:BackgroundIonizing radiation (IR) in combination with microtubule stabilizing agents (MSA) is a promising combined treatment modality. Supra-additive treatment responses might result from direct tumor cell killing and cooperative indirect, tumor cell-mediated effects on the tumor microenvironment. Here we investigated deregulation of matrix metalloproteinase (MMP) activity, as an important component of the tumor microenvironment, by the combined treatment modality of IR with the clinically relevant MSA patupilone.MethodsExpression, secretion and activity of MMPs and related tissue inhibitors of metalloproteinases (TIMPs) were determined in cell extracts and conditioned media derived from human fibrosarcoma HT1080 and human glioblastoma U251 tumor cells in response to treatment with IR and the MSA patupilone. Treatment-dependent changes of the invasive capacities of these tumor cell lines were analysed using a Transwell invasion assay. Control experiments were performed using TIMP-directed siRNA and TIMP-directed inhibitory antibodies.ResultsEnzymatic activity of secreted MMPs was determined after treatment with patupilone and irradiation in the human fibrosarcoma HT1080 and the human glioblastoma U251 tumor cell line. IR enhanced the activity of secreted MMPs up to 2-fold and cellular pretreatment with low dose patupilone (0.05-0.2 nM) counteracted specifically the IR-induced MMP activity. The cell invasive capacity of HT1080 and U251 cells was increased after irradiation with 2 Gy by 30% and 50%, respectively, and patupilone treatment completely abrogated IR-induced cell invasion. Patupilone did not alter the level of MMP expression, but interestingly, the protein level of secreted TIMP-1 and TIMP-2 was lower after combined treatment than after irradiation treatment alone. Furthermore, siRNA depletion of TIMP-1 or TIMP-2 prevented IR-mediated induction of MMP activity and cell invasion.ConclusionsThese results indicate that patupilone counteracts an IR-induced MMP activation process by the reduction of secreted TIMP-1 and TIMP-2 proteins, which are required for activation of MMPs. Since IR-induced MMP activity could contribute to tumor progression, treatment combination of IR with patupilone might be of great clinical benefit for tumor therapy.

Dataset Information

Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by the membrane-anchored glycoprotein RECK.

Publications

Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by the membrane-anchored glycoprotein RECK.

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