Carcinogenesis vol.29 no.11 pp.2236–2242, 2008 doi:10.1093/carcin/bgn204 Advance Access publication August 27, 2008 Reduction of brain metastases in plasminogen activator inhibitor-1-deficient mice with transgenic ocular tumors C.M.Maillard1,Ã, C.Bouquet2, M.M.Petitjean1, the urokinase-type plasminogen activator (uPA) and tissue-type plas- M.Mestdagt1, E.Frau2, M.Jost1, A.M.Masset1, minogen activator (tPA) (for review, see refs 1–3). Both uPA and tPA P.H.Opolon2, F.Beermann4, M.M.Abitbol3, J.M.Foidart1,5, are inhibited by several serine protease inhibitors (Serpins) including M.J.Perricaudet2 and A.C.Noe¨l1 plasminogen activator inhibitor (PAI)-1, PAI-2, PAI-3 (protein C in- activator), protease nexin-1 and mammary serine protease inhibitor 1Laboratory of Tumor and Development Biology, GIGA-Cancer, Tour de (Maspin) (1,4). In addition to its inhibitory role during PA-mediated Pathologie (B23), University of Lie`ge, Sart-Tilman, B-4000 Lie`ge, Belgium, proteolysis, PAI-1 also interacts with vitronectin involved in cell adhe- 2 CNRS UMR 8121 Univ Paris Sud, Vectorologie et Transfert de Ge`nes, sion and migration (5,6), and low-density lipoprotein receptor-related Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, France, protein implicated in the endocytosis of cell surface molecules (7,8). Downloaded from https://academic.oup.com/carcin/article/29/11/2236/2476749 by guest on 27 September 2021 3 # Centre d e´tudes et de Recherches The´rapeutiques en Ophtalmologie, Faculte´ As an inhibitor of uPA, PAI-1 was long thought to be an inhibitor of de Me´decine Necker, 156 rue de Vaugirard, 75015 Paris, France, 4Swiss Institute for Experimental Cancer Research (ISREC), Faculty of life Sciences, tumorigenesis and angiogenesis. A high level of PAI-1 protein in Ecole Polytechnique Fe´de´rable de Lausanne (EPFL), Chemin de Boveresses extracts of human primary malignant tumors is one of the most in- 155, CH-1066 Epalinges, Switzerland and 5Department of Gynecology and formative biochemical markers of a poor prognosis in several human Obstetrics, CHU, B-4000 Lie`ge, Belgium cancer types (2,3,9). A dose-dependent effect of PAI-1 on patholog- ical angiogenesis has been observed both in vitro (10) and in vivo ÃTo whom correspondence should be addressed. Laboratory of Tumor Biology and Development, Institute of Pathology CHU-B23, University of Lie`ge, Sart- (11–14). Tumor growth and angiogenesis are impaired in mice with Tilman, B-4000 Lie`ge, Belgium. Tel: þ32 4 366 25 69; Fax: þ32 4 366 29 36; a PAI-1 gene deletion (15–17) or following the induction of PAI-1 Email: [email protected] expression at supraphysiological levels by adenoviral gene transfer or Correspondence may also be addressed to A.C.No¨el. tumor cell transfection, the administration of recombinant PAI-1 pro- Email: [email protected] tein and in transgenic mice overexpressing PAI-1 (12,14,18–20). Plasminogen activator inhibitor-1 is known to play a paradoxical The impact of PAI-1 on metastatic dissemination is less clear. An positive role in tumor angiogenesis, but its contribution to metastatic increase in pulmonary metastasis has often been observed in response spread remains unclear. We studied the impact of plasminogen ac- to PAI-1 administration (21,22). However, other studies have reported tivator inhibitor (PAI)-1 deficiency in a transgenic mouse model of no difference in metastatic dissemination with PAI-1 level (23,24). It ocular tumors originating from retinal epithelial cells and leading should be pointed out that these data obtained after induction of ex- to brain metastasis (TRP-1/SV40 Tag mice). PAI-1 deficiency did not perimental tumors by massive tumor cell injection may not be repre- affect primary tumor growth or vascularization, but was associated sentative of events in the pathogenesis of real cancers. Many of the with a smaller number of brain metastases. Brain metastases were models used to study metastasis bypass the early events of tumor found to be differentially distributed between the two genotypes. progression toward a metastatic stage. Genetically engineered mouse PAI-1-deficient mice displayed mostly secondary foci expanding models that spontaneously develop cancer in a target organ may thus from local optic nerve infiltration, whereas wild-type animals dis- provide a powerful tool for more accurately mimicking the natural played more disseminated nodules in the scissura and meningeal history of cancer. A single study has evaluated the impact of PAI-1 spaces. SuperArray GEarray analyses aimed at detecting mole- deficiency in a transgenic mouse model of cancer (24). Primary breast cules potentially compensating for PAI-1 deficiency demonstrated tumor development and lung metastasis were similar in MMTV-PymT an increase in fibroblast growth factor-1 (FGF-1) gene expression mice with and without PAI-1 expression (24). It remains unclear in primary tumors, which was confirmed by reverse transcription– whether possible compensatory or redundant mechanisms can mediate polymerase chain reaction and western blotting. Our data provide tumor angiogenesis and metastatic dissemination in the absence of the first evidence of a key role for PAI-1 in a spontaneous model of PAI-1. The aim of this paper is to evaluate the role of PAI-1 on tumor metastasis and suggest that angiogenic factors, such as FGF-1, may metastasis and to search for putative factors that could substitute for be important for primary tumor growth and may compensate for PAI-1 in genetically induced tumors. We backcrossed PAI-1-deficient the absence of PAI-1. They identify PAI-1 and FGF-1 as important mice with TRP-1/SV40 Tag transgenic mice, which spontaneously targets for combined antitumor strategies. develop ocular tumors derived from retinal pigmented epithelium (RPE), leading to brain metastasis (25). While PAI-1 deficiency has no effect on primary ocular tumor growth, it reduces the number of metastatic foci and affects their distribution in the brain. The present paper reports for the first time that PAI-1 contributes to the early steps Introduction of metastatic dissemination in a transgenic mouse tumor model re- Angiogenesis—the formation of new blood vessels—is an important capitulating all the steps of metastasis. Attempts to identify molecules rate-limiting step during tumor growth and metastatic dissemination. potentially compensating for PAI-1 deficiency, we have demonstrated This process requires the co-ordinated regulation of adhesive, pro- an increase in levels of messenger RNA (mRNA) and protein for teolytic and migrating events involving different proteolytic systems. fibroblast growth factor-1 (FGF-1) in primary tumors. Many clinical studies have established a correlation between adverse outcome in patients with multiple cancer types and high levels of Materials and methods serine proteases of the plasminogen activator (PA) system such as Genetically modified mice Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; FGF-1, TRP-1/SV40 Tag transgenic mice were generated by insertion into Y chromo- fibroblast growth factor-1; KO, knockout; Maspin, mammary serine protease some of a 1.4 kb fragment of tyrosine-related protein 1 (TRP-1) promoter fused to inhibitor; MMP, matrix metalloproteinase; mRNA, messenger RNA; PA, plas- SV40 Tag transforming sequence (25). Female PAI-1-deficient mice (PAI-1À/À) minogen activator; PAI, plasminogen activator inhibitor; RPE, retinal pig- (15) in C57BL/6J background were mated with male TRP-1/SV40 Tag mented epithelium; RT–PCR, reverse transcription–polymerase chain transgenic mice. These TRP-1/SV40 Tag PAI-1þ/À males were backcrossed reaction; Serpin, serine protease inhibitors; TIMP, tissue inhibitor of metal- to C57BL/6J for six generations. TRP-1/SV40 Tag PAI-1À/À mice were obtained loprotease; tPA, tissue-type plasminogen activator; TRP-1, tyrosine-related by breeding these males to PAI-1À/À females or PAI-1þ/þ [wild-type (WT)] protein 1; uPA, urokinase-type plasminogen activator; VEGF, vascular endo- female littermates. Mouse experimentation was done in accordance to the guide- thelial growth factor; WT, wild-type. lines of the University of Lie`ge regarding the care and use of laboratory animals. Ó The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 2236 PAI-1 in ocular tumor and metastasis Tissue sample collection and brain metastasis analysis Reverse transcription–polymerase chain reaction analysis For histological analysis, animals were killed 64 days after birth. The entire Reverse transcription–polymerase chain reaction (RT–PCR) amplification was head of each animal was fixed in 4% formalin. After overnight decalcification carried out with the GeneAmp Thermostable rTth reverse transcriptase RNA (Sakura Finetek, Zoeterwoude, The Netherlands), tissue samples were rinsed PCR kit (Perkin Elmer Life Sciences, Boston, MA) with specific pairs of primers in water for 1 h and incubated in 4% formalin for 1 h. Five frontal fragments of (Table I). RT–PCR products were resolved by electrophoresis in 10% poly- the head were cut to isolate several brain areas covering the entire organ. acrylamide gels and analyzed with a Fluor-S MultiImager after staining with Paraffin sections cut from each head fragment (five per animal) were stained Gelstar dye (FMC BioProducts, Heidelberg, Germany). RT–PCR products were with hematoxylin and eosin. Total number of metastases per mouse was de- quantified by normalization with respect to 28S ribosomal ribonucleic acid. termined as the number of metastatic foci on five sections for each animal (n 5 22 for PAI-1À/À and n 5 19 for WT mice). Incidence of metastasis was Western blotting calculated as the percentage of mice with one or more metastatic nodules in the Entire eyes collected on day 64 were placed in lysis tubes (MagNA Lyser brain. Metastasis severity was scored as follows: minimal (score 0 5 no met- Green Beads, Roche Diagnostics) containing 300 ll of lysis buffer (50 mM astatic nodule), medium (score 1 5,4 metastatic nodules) or extensive in- Tris–HCl, pH 7.5; 110 mM NaCl; 10 mM ethylenediaminetetraacetic acid; volvement (score 2 5 5–7 metastatic nodules).