Targeting the Tumor Microenvironment with Chemically Modified Tetracyclines: Inhibition of Laminin 5 2 Chain Promigratory Fragments and Vasculogenic Mimicry

The laminin 5 (Ln-5) 2 chain and matrix metalloproteinases (MMPs) MMP-2 and membrane type 1 (MT1)-MMP act cooperatively and are required for highly aggressive melanoma cells to engage in vasculogenic mimicry when cultured on a threedimensional matrix. Furthermore, generation of Ln-5 2 chain promigratory fragments by MMP-2 and MT1MMP proteolysis is necessary for an aggressive tumor cell-preconditioned matrix to induce vasculogenic mimicry in poorly aggressive tumor cells. These observations suggest that treatment regimes that specifically target aggressive tumor cells may fail to take into account changes in the extracellular microenvironment that persist after removal or destruction of an aggressive tumor and could result in a recurrence or continuance of the tumor. As a potential therapeutic approach to address this concern, the work presented here measured the molecular consequences of adding a chemically modified tetracycline (CMT-3; COL-3) that inhibits MMP activity to aggressive metastatic melanoma cells in three-dimensional culture. COL-3 inhibited vasculogenic mimicry and the expression of vasculogenic mimicry-associated genes in aggressive cells, as well as the induction of vasculogenic mimicry in poorly aggressive cells seeded onto an aggressive cell-preconditioned matrix. Furthermore, molecular analysis revealed that COL-3 not only inhibited the generation of Ln-5 2 chain promigratory fragments in the aggressive cell-preconditioned matrix but also inhibited the induction of Ln-5 2 chain gene expression in poorly aggressive cells by the aggressive cell-preconditioned matrix. These results suggest that COL-3 (and related chemically modified tetracyclines) may be useful in targeting molecular cues in the microenvironment of aggressive tumors and could potentially be used in a combinatorial manner with other therapies that specifically target and kill aggressive tumor cells. Introduction The Ln-5 2 chain, MMP-2, and MT1-MMP act cooperatively and are required for highly aggressive melanoma tumor cells to engage in vasculogenic mimicry when cultured on a three-dimensional ECM (1). Furthermore, Ln-5 2 chain promigratory fragments generated in an aggressive tumor cell-preconditioned matrix by MMP-2 and MT1-MMP proteolysis induce vasculogenic mimicry in poorly aggressive melanoma cells seeded onto this matrix (1). These observations suggest that deposition of Ln-5 2 chain into an ECM by aggressive melanoma cells can act as a latent trigger for aggressive behavior, and less aggressive cells entering this preconditioned environment (in the presence of active MMP-2 and MT1-MMP) respond by assuming a more aggressive, vasculogenic mimicry phenotype. As a result, treatment regimens that specifically target aggressive tumor cells may fail to address changes in the ECM microenvironment that persist after removal or destruction of the aggressive cells and could result in a recurrence or continuance of the tumor. Therefore, conceptually, a therapeutic regime that targets aggressive tumor cells should also consider changes in the microenvironment of the tumor that may ultimately harbor molecular cues or triggers that can induce less aggressive cells to become more aggressive, even after the aggressive cells have been removed or killed. A key component for developing this type of therapeutic approach might involve inhibiting the generation of Ln-5 2 chain promigratory fragments in the ECM of aggressive tumors to produce a more benign and less inductive microenvironment. One class of drugs that is presently in clinical trials based on their antimetastatic (2–5) and antiangiogenic (6, 7) activities is the CMTs. CMTs are potent inhibitors of MMP activity (2–7), and COL-3 (6-demethyl-6-deoxy-4-dedimethylaminotetracylcine) is currently being evaluated for treatment of patients with refractory solid tumors (8–10). The work described in this study presents evidence that COL-3 treatment not only inhibits vasculogenic mimicry by aggressive/metaReceived 7/10/02; revised 9/23/02; accepted 9/30/02. 1 Supported by NIH/National Cancer Institute Grants CA83137 (to R. E. B. S.), CA80318, CA88043-02S1, and CA59702 (to M. J. C. H.). 2 To whom requests for reprints should be addressed, at Department of Anatomy and Cell Biology, The University of Iowa, 51 Newton Road, 1-100 BSB, Iowa City, IA 52242-1109. Phone: (319) 335-7755; Fax: (319) 3357770; E-mail: [email protected]. 3 The abbreviations used are: Ln-5, laminin 5; CMT, chemically modified tetracycline; ECM, extracellular matrix; MMP, matrix metalloproteinase; MT1-MMP, membrane type 1-MMP; RT-PCR, reverse transcription-PCR; VEGF, vascular endothelial growth factor; VE-cadherin, vascular endothelial-cadherin; TIE-1, tyrosine kinase with immunoglobulin and epidermal growth factor homology domains; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 1173 Vol. 1, 1173–1179, November 2002 Molecular Cancer Therapeutics on October 28, 2017. © 2002 American Association for Cancer Research. mct.aacrjournals.org Downloaded from static human melanoma cells grown in three-dimensional culture but also prevents the production of Ln-5 2 chain promigratory fragments in the ECM and blocks the induction of vasculogenic mimicry by poorly aggressive melanoma cells placed on the aggressive cell-preconditioned matrix. These results suggest that COL-3 (and related CMTs) may provide an effective therapy for targeting molecular cues in the tumor microenvironment that could be administered in a coordinated manner with other therapies that specifically target and kill aggressive tumor cells. Materials and Methods Cell Culture. The human cutaneous (C8161) and uveal (MUM-2B and MUM-2C) melanoma cell lines have been described previously (1, 11–15) and were maintained in RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and 0.1% gentamicin sulfate (Gemini Bioproducts, Calabasas, CA). Cell cultures were determined to be free of Mycoplasma contamination using the GenProbe rapid detection system (Fisher, Itasca, IL). A critical aspect of this work was the careful production of three-dimensional type I collagen gels (1) as follows: 25 l of type I collagen (average 3 mg/ml; Collaborative Biomedical, Bedford, MA) were dropped onto 18-mm glass coverslips in 12-well culture dishes and polymerized with an application of 100% ethanol for 5 min at room temperature. After washing with PBS (minus divalent cations), tumor cells were seeded onto the three-dimensional gel in complete medium. For experiments designed to analyze the ability of the cells to engage in vasculogenic mimicry using phase-contrast microscopy, cells were plated onto the three-dimensional matrix with or without 3 g/ml COL-3 or CMT-5 (kind gifts from CollaGenex Pharmaceuticals, Inc., Newton, PA) at the time of seeding and daily thereafter. The cultures were then observed after 4 days, and images were captured digitally using a Zeiss Televal inverted microscope (Carl Zeiss, Inc., Thornwood, NY) and Hitachi HV-C20 charge-coupled device camera (Hitachi Denshi America, Ltd., Woodbury, NY). For experiments designed to analyze the ability of the poorly aggressive MUM-2C cells to engage in vasculogenic mimicry when placed on a matrix preconditioned by the highly aggressive MUM-2B cells, the MUM-2B cells were removed after 3 days with 20 mM NH4OH followed by three quick washes with water, PBS, and then complete medium. Western Blot and Substrate-incorporated SDS-PAGE (Zymographic) Analyses. Detection of the Ln-5 2 chain and its cleavage fragments in three-dimensional cultures containing highly aggressive C8161 or MUM-2B cells plus and minus either COL-3 or CMT-5 was performed four times independently as follows: cells plus and minus 3 g/ml COL-3 or CMT-5 were seeded as stated above in 12-well culture dishes with the CMT added at the time of seeding and daily thereafter. After 3 days, the cultures were washed with PBS, and the total volume of each well was scraped into 50 l of sample buffer (plus -mercaptoethanol), sonicated for 2–4 s, and microfuged at 4°C for 10 min, and the supernatant was heated at 95°C for 5 min. Protein concentrations were determined using a bicinchoninic acid assay (Pierce, Rockford, IL) and 7.5% SDS-PAGE gels loaded with 100 g protein/lane normalized to the same (10 l) volume with sample buffer. After electrophoresis, the separated proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA), and the Ln-5 2 chain and its fragments were subsequently detected using antibody D4B5 (Chemicon, Temecula, CA), a secondary antibody conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratories, West Grove, PA) and enhanced chemiluminescence (ECL; Amersham, Piscataway, NJ). Samples were prepared for zymography three independent times as follows: cells were seeded onto the collagen three-dimensional matrix in the presence of 3 g/ml COL-3 or CMT-5 (added daily thereafter) or no CMT in serum-free medium containing Mito (Mito is a fully defined concentrate composed of hormones, growth factors, and other physiological components required for the optimum propagation of cultured cells under reduced or short-term serumfree conditions; Collaborative Biomedical). After 2 days, the supernatants were harvested and microfuged to remove nonadherent cells and debris, and the supernatant was added 2 parts to 1 part sample buffer without -mercaptoethanol or heating. Equal volumes of sample were then electrophoresed in 10% SDS-polyacrylamide gels containing 0.1% gelatin as described previously (2, 16). After processing and staining, the resulting zymogram was digitized, the areas of the cleared zones associated with MMP activity were determined using Scion Image for Windows (Beta 4.0.2; Scion Corp.), and the values were calculated relative to the control samples normalized to a value of 1.00. Semiquantitative RT-PCR Analysis. Total RNA from untreated and CMT-treated melanoma cell lines was prepared using Trizol reagent (Invitrogen) as per the manufacturer’s protocol and reverse transcribed using the Advantage PCR kit as per the manufacturer’s instructions (BD Clontech Laboratories, Palo Alto, CA). PCR amplifications were performed with gene-specific primers (Table 1) with annealing temperature and number of amplification cycles optimized using cDNA from untreated MUM-2B and MUM-2C cells. PCR amplification reactions were performed in a Robocycler Infinity Thermocycler (Stratagene, La Jolla, CA) as follows: 1 cycle of 94°C for 1 min; 25–30 cycles of 94°C for 1 min, 68°C for 2.5 min, and 72°C for 1 min; and 1 cycle of 72°C for 5 min. GAPDH primers (BD Clontech Laboratories) were used as controls for PCR amplification, and PCR fragments were ligated into the pCR2.1 TOPO sequencing vector as per the manufacturer’s instructions (Invitrogen). Plasmid DNA was isolated and subjected to DNA sequencing analysis using fluorescent Sanger-based dideoxy sequencing on a PEApplied Biosystems Model 3700 Automated Sequencer (University of Iowa DNA Facility). Two plasmids from each primer set were sequenced and shown to have 100% identity to the expected DNA sequence. The RT-PCRs were performed three times with independently derived samples.