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Overexpression of Plasminogen Activator Inhibitor 2 in Human Melanoma Cells Inhibits Spontaneous Metastasis in Scid/Scid Mice BARBARA M

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Overexpression of Plasminogen Activator Inhibitor 2 in Human Melanoma Cells Inhibits Spontaneous Metastasis in Scid/Scid Mice BARBARA M Proc. Natl. Acad. Sci. USA Vol. 92, pp. 205-209, January 1995 Medical Sciences Overexpression of plasminogen activator inhibitor 2 in human melanoma cells inhibits spontaneous metastasis in scid/scid mice BARBARA M. MUELLER*t, YAN BIN YUt, AND WALTER E. LAUGt§ *Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037; tDepartment of Pediatrics, Division of Hematology-Oncology, Children's Hospital of Los Angeles, University of Southern California School of Medicine, Los Angeles, CA 90027; and §Division of Biology, California Institute of Technology, Pasadena, CA 91125 Communicated by M. Frederick Hawthorne, University of California, Los Angeles, CA, September 19, 1994 ABSTRACT A metastatic human melanoma cell line that invasion of human tumors grown s.c. in mice (8), and exper- produces urokinase-type plasminogen activator was stably trans- imental metastasis of murine melanoma cells in mice (9). fected with cDNA encoding human plasminogen activator inhibitor Overexpression of uPA in poorly metastatic murine melanoma 2 (PAI-2). Transfected clones expressed PAI-2 at levels two to nine cells (10) or in H-ras-transformed NIH 3T3 fibroblasts (11) times higher than both the parental cell line and mock transfec- enabled these cells to form experimental metastases. Overex- tants, as detected by ELISAofcell lysates and conditioned medium. pression of enzymatically inactive uPA reduced metastasis of The clone with the highest PAI-2 expression exhibited complete prostate carcinoma cells (12). inhibition ofsoluble and cell-surface-bound plasminogen activator We previously described the highly metastatic human mel- activity. The level ofPAI-2 overexpression in these clonal cell lines anoma cell line M24met (13) that secretes a spectrum of correlated positively with the inhibition of their ability to degrade proteolytic enzymes, including type IV collagenases, intersti- extracellular matrix in vitro. Parental, mock-transfected, and PAI- tial collagenase, and uPA (14). In vitro, this cell line degrades 2-transfected cell lines produced rapidly growing tumors when interstial and basement membrane matrices with uPA- injected s.c. into the skin of mice with severe combined immuno- dependent removal of matrix glycoprotein as a prerequisite deficiency. The tumors producing the highest levels of PAI-2 were step preceding metalloproteinase-dependent collagenolysis. surrounded by a dense tumor capsule. Both parental cells and Consequently in this model the addition of exogenous recom- mock-transfected cells metastasized from s.c. tumors to binant PAI-2 inhibits dissolution of all matrix components invariably (14). Here, we address the role of PA activity in human lymph nodes and lungs of mice. PAI-2-transfected cell lines pro- melanoma metastasis in severe combined immunodeficient duced significantly less or no metastases. Taken together, these (SCID) mice. We stably transfected our metastatic melanoma data indicate a critical role for plasminogen activator activity in cell line with full-length PAI-2 cDNA and analyzed the melanoma invasion and metastasis. resulting transfectants for their ability to degrade ECM and to metastasize in SCID mice from s.c. tumors to distant organs, Invasion of surrounding tissues and metastasis to distant such as lymph nodes and lungs. organs are the main clinical characteristics of malignant tu- mors. The pathobiological mechanisms enabling tumor cells to MATERIALS AND METHODS invade through occluding extracellular matrix (ECM) and to disseminate to distant organs remain to be elucidated. Various Cell Culture. The human melanoma cell line M24met has proteolytic enzymes elaborated by tumor cells have been been described (13). For this study M24met cells were sub- implicated as important in invasion and metastasis (1, 2). cloned by limiting dilution, and a clonal line designated Plasminogen activator (PA) and plasmin play a central role M24met/cl was established. M24met/cl cells were grown in in tissue degradation and remodeling (3). Plasmin, the active RPMI 1640 medium 10% fetal bovine serum/2 mM glutamine. form of plasminogen, degrades ECM components such as Transfection. The full-length cDNA encoding for human and fibrin and also mediates activation of PAI-2 was from E. K. 0. Kruithof (Lausanne, Switzerland). It fibronectin, laminin, was cloned into the unique EcoRI restriction site of the certain metalloproteinases. Two distinct activators of plasmin- expression vector pEMSVSCRIBE-a2 (6) by using specific ogen are known, urokinase-type PA (uPA) and tissue-type PA, adaptors. M24met/cl cells were cotransfected with the simian which are tightly controlled by specific PA inhibitors (PAls) virus 40-driven PAI-2 expression vector and pSV2-neo with the namely, PAI-1 and PAI-2. The fine-tuned balance between PA lipofectin reagent (GIBCO/BRL). Stable transfectants were and PAI activities is responsible for controlled pericellular isolated by neomycin selection (500 ,tg/ml). Mock transfec- proteolysis and appears to be lacking in tumor cells, resulting tants were obtained under identical conditions using pSV2-neo in continuous, uncontrolled proteolytic breakdown of the and pEMSVSCRIBE-a2 without insert. PAI-2-expressing ECM by these cells, leading to tissue invasion and ultimately clones were identified in a fibrin agarose screening assay, as metastasis. Restoration of this balance by inhibition of tumor described (6). PAI-2 antigen in conditioned medium (CM) and cell-derived PA activity should therefore result in suppression cell lysates was quantified by using the Imubind PAI-2 ELISA of invasion and metastasis. kit (ADI, Greenwich, CT). Thus, inhibition of uPA by specific inhibitors or anticatalytic PA and PAI-2 Activity. Cells (105/35-mm dish) were kept in antibodies resulted in inhibition of tumor cell invasion and serum-free medium for 16 hr. PA and PAI activities in cell CM metastasis. PAI-1 and PAI-2 were shown to inhibit ECM and eluates (10-fold concentrated) were determined in 5-,ul degradation by human tumor cell lines in vitro (4-6). Anti-uPA aliquots separated by SDS/PAGE by regular or reverse fibrin- antibodies inhibited invasion and metastasis of a human squamous carcinoma cell line in chicken embryos (7), local Abbreviations: ECM, extracellular matrix; PA, plasminogen activator; uPA, urokinase-type PA; PAI, PA inhibitor; uPAR, uPA receptor; SCID, severe combined immunodeficiency; CM, conditioned medium. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" in Immunology, IMM13, The Scripps Research Institute, 10666 North accordance with 18 U.S.C. §1734 solely to indicate this fact. Torrey Pines Road, La Jolla, CA 92037. 205 206 Medical Sciences: Mueller et al. Proc- Natt Acad Sci USA 92 (1995) agarose zymography (15). SDS/PAGE gels were washed in Table 1. PAI-2 protein in melanoma cell lysates and CM 2.5% Triton X-100 and underlaid with a gel consisting of PAI-2, ng/106 cells bovine fibrinogen (10 mg/ml), 2.5% low-temperature-geling agarose, thrombin, and plasminogen and, for reverse zymog- Cell line Cell lysate CM raphy only, also uPA. Zones of lysis were evaluated after 8- to M24met/cl 62 ± 2 122 ± 40 12-hr incubation at 37°C. M24met/cl/mock 1 62 ± 23 155 ± 21 Immunoblotting. Serum-free CM, neutralized eluates, or PAI-2-2 187 ± 26 413 ± 56 cell lysates were generated, as described (16), separated on PAI-2-4 290 ± 41 1310 ± 125 SDS/10% PAGE, and electroblotted onto nitrocellulose PAI-2-6 222 ± 30 389 ± 66 membranes. Primary antibodies were goat anti-human uPA, PAI-2-13 200 ± 22 442 ± 7 rabbit anti-human uPA receptor (uPAR) (ADI), and rabbit PAI-2-25 182 ± 3 244 ± 33 anti-human PAI-2 antiserum (E. K. 0. Kruithof). PAI-2-35 191 ± 38 759 ± 82 Matrix Degradation. Degradation of radiolabeled ECM produced by R22C1D rat smooth muscle cells in vitro was Data are reported as mean ± SD for quadruplicate samples in PAI-2 determined as described (14). Cells (2 x 105/35-mm dish) in ELISA. complete tissue culture medium were seeded onto the matri- Soluble uPA was demonstrated for all cell lines tested (Fig. 2A). ces. Radioactivity released into the supernatant was deter- A nonspecific increase in uPA expression upon stable transfec- mined at the times indicated, and cumulative counts were plotted after subtraction of counts released by medium alone. tion has been observed earlier (6). It was notable that all After 13 days tumor cells were lysed with 0.025 M (NH4)OH, transfected cell lines produced more uPA than the parental cell and the residual matrices were analyzed by sequential enzyme line. Because the cells were grown in serum-free medium-i.e., in treatment (14). the absence of plasmin, most uPA is present in the enzymatically Spontaneous Metastasis Assay. Tumor cells (2 x 106 in 100 inactive proform, thus escaping detection by fibrin overlay. PAI-2 ,ll of phosphate-buffered saline) were injected s.c. into the was detectable by immunoblotting only in the supernatant ofcells flank of female, 6- to 8-week-old CB1.7 scid/scid mice. Tumors transfected to overexpress PAI-2. Distinct bands of surface- were measured with calipers, and volumes were calculated as bound uPA (52 kDa) were detected in such eluates from all cell (a2xb)/2, where a is the width and b is the tumor length. When lines (Fig. 2B). In addition, the anti-uPA antibody stained uPA- PAI complexes (102 kDa) from the surface of the PAI-2- the tumors reached a volume of - 1000 mm3, usually on day 15 after injection, they were fixed in 10% buffered formalin and transfected cell lines. Anti-PAI-2 antibodies detected a glycosy- stained with Gomori's trichrome stain to visualize collagen lated and a nonglycosylated form of PAI-2 (59 and 47 kDa, fibers and tumor capsules. respectively), as well as PA-PAI-2 complexes on the surface of Twenty-one days after removal of the primary tumor, mice PAI-2-transfected cells (Fig.
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