- Email: [email protected]
86 Reduction of tumor cell migration and metastasis by plasminogen activator inhibitor gene transfer
SESSION IX: Physiology and Disease
84
85
ROLE OF MATRIX DEGRADING PROTEINASES IN WOUND HEALING AND SKIN CANCER. Leif R. Lond t, John Romer j, Thomas H. Bugge 1'2, Ken Brown 3, Chnng-Hyun Lee Sogaard I, Jay Degen z and Keld Dana t IFinsen Laboratory, Rigshospitalet, DK-2100 Copenhagen, Denmark, 2Childrens Hospital Research Foundation, Cincinatti, Ohio, USA, JBeatson Institute, Glasgow, United Kingdom Prnteolytic degradation of the extracellular matrix is a central aspect of normal and pathological tissue remodeling events in the skin. By in situ hybridization we find expression of both uPA and uPAR mRNA in the invading cancer cells in human squamons cell skin carcinomas, and during mouse skin wound healing mRNA for uPA and uPAR is expressed by the migrating kemtinocytes, suggesting that the skin cancer cells are imitating these keratinocytes. Following treatment of mouse skin with tumor promoting phorbol esters, a fast induction ofuPA mRNA is seen both in the dermis and epidermis, supporting the notion for an active role of the PAsystem during skin tumor promotion. During skin carcinogenesis induced by DMBA/PMA treatment or by transgane expression of the Rns oncogene under control of the Keratin 5 promoter, only uPA is expressed by papillomas, while both uPA and uPAR are expressed after the transition from papillomas to carcinoma. To investigate the role of plasminogen activation in skin tumor formation, skin tumors were induced in plasminogen-deficient mice by breeding into the transgene Ras expressing mice. Preliminary results show no significant difference between Rasexpressing Pig(4") mice and Pig(+/*) littermate control mice in the type, rate and frequency of skin tumor development. However, the carcinomas showed an abnormal histology with increased extracellular matrix deposition and cell death in the plasminogen-deficient mice compared to the litter mate controls. We have previously reported that skin wound healing is delayed in plasminogen-deficient mice, but also that the wounds eventually will heal. By in situ hybridization we find an expression pattern of the matrix metalloprotcinases gelatinasc B, collagenase-3 and stromeiysin-1 in keratinecytes at the leading edge of epithelial outgrowths similar to that ofuPA and uPAR. These findings suggest that other proteinases are able to substitute for the plasminogen activation system, thereby providing a functional overlap.Thns we have started breeding mice deficient for either gelatinase B or stromelysin-I with plasminogen deficient mice in order to elucidate potential functional overlaps during skin wound healing and skin carcinogenesis.
INHIBITION OF IN VIVO TUMOR GROWTH BY MURINE AND HUMAN UROKINASE RECEPTOR ANTAGONISTS.
86 R E D U C T I O N OF T U M O R C E L L M I G R A T I O N A N D M E T A S T A S I S BY P L A S M I N O G E N A C T I V A T O R INHIBITOR GENE T R A N S F E R M. Prans, K. Wanteriekx, D. Collen and R. D. Gerard Center for Transgene Technology and Gene Therapy, Vlaams lnteruniversitair lnstituut voor Biotechnologie, KU Leuven, Herestraat 49, B-3000 Leuven, Belgie. Urokinase (u-PA), its cellular receptor (u-PAR) and other components of the fibfinolytic cascade have been implicated in various biological processes including tissue remodeling, tumor growth and metastasis. There is a direct correlation between the invasiveness of tumor cells and the level of expression of u-PA, u-PAR and plasminogen activator inhibitor 1 (PAL1). Adanoviral gene transfer has been shown to be a powerful tool for the expression of foreign genes in somatic cells. The aim of this study was to use adenoviral gene transfer of components of the fibrinolytic system to modulate the migratory behavior of human HT-1080 fibrosarcoma/n vitro and the metastatic behavior of HT-1080 ceils in vivo by gene transfer of PAI- 1 and PAI-2. In an in vitro assay using Transwell inserts coated with Matrigel, there was a positive correlation (r= 0.83) between u-PA secretion and tumor cell migration. Migration could be augmented 50% (p=0.027) by adenovirus-mediatud overexpression of u-PA and reduced 30% (p--0.027) by overexpression of PAI-1. Tumor metastasis was studied in vivo in nude mice using two routes of gene transfer. In the first, HT-1080 cells stably expressing luciferase were directly infected with recombinant adenoviruses expressing PAI-1, PAI-2 or control virus and then implanted subcutaneously. Metastasis was quantified at 26 days by assaying luciferase in organ homogenates. Overexpression of PAI-1 and PAI-2 in the tumor cells reduced the percentage of animals with detectable metastasis to the lung by 50% (p-=0.0098) and reduced the burden of metastasis 4-fold (p-~.004 for PAI-1 and p=0.003 for PAI-2). Both tumor incidence and tumor burden in other organs were not significantly affected by PAI gene transfer. In the second, uninfected tumor cells were implanted and liver gene transfer for systemic overexpression of PAI-1 and PAI-2 was performed by tall vein injection of 5x10 8 infectious units of adenovirus. In contrast to the direct infection experiments, overexpressinn of PAI-1 or PAI-2 in the circulation had no significant effect on the incidence of metastasis. PAI-1 failed to achieve significant reduction of the metastatic burden to any organ. However, PAI-2 gene transfer reduced the tumor burden in the brain and lung 6-fold (p<0.01). These results suggest that u-PA activation of plasminogen is involved in the metastasis of human fibrosarcoma cells, that adenoviral gene transfer can modulate both tumor cell migration and metastasis and that the site of expression is critical for the biological activity of PAI-1.
Steven Resenberg, Laura V. Doyle, Robert J. Drummond, Nathalie Dubois-Stringfellow, Hye Yeeng Min, Patriee Pitut, Robert Tressler, Jennifer R. Strattnn-Thomas, and Catherine Zandnnella, Chiron Corporation, 4560 Hortnn Street, Emeryvine, CA 94608. The urokinase: urokinase receptor system has been implicated in tumor growth, angiogenesis, and metastasis, and further has been correlated with progression of a variety of human tumors. However, the lack of a significant phenotype in the uPAR deficient mice has led to significant skepticism on the role of uPAR in in vivo processes. We have expressed and purified antagonists of the routine and human receptors from insect or yeast cells (Min et al., Cancer Res. 56,2428-2433, 1996; Stratton-Thomas etal.,Prot.Eng. 8,463-470, 1995). These antagonists consist of the EGFlike domain of the urokinases, or the domains fused to the constant region of human IgG1. The effects of these molecules on tumor has been tested in 2 models in immnnocomprisad mice: M24Met, human melanoma, and MDA-MB-231, mammary carcinoma, which express human uPAR in vitj..~o, with the highest level found on M24Mct. Dramatic reductions in tumor growth in SCID mice were observed for the M24Met cell line after treatment with hl-48Ig, despite a lack of effeet on the growth of the cell line in vitro. However, in SCID mice lacking complement and natural killer cells, no effect was observed, suggesting the hl-481g was acting as a marker for complement/NK mediated tumor cell killing. In contrast, MDA-MB231 was significantly inhibited by hl-48, and by murine 1-48Ig in SCID/C/NK deficient mice. These results support a previous report .in which expression of the PA system on both tumor cells and stromal cells has been observed for the MDA-MB-231 system (Romer et al., Int. J. Cancer 57, 553-560, 1994), and show that the PA system plays a functional role on both tumor cells and stromal cells in facilitating tumor growth in this in vivo model.
25
84
85
ROLE OF MATRIX DEGRADING PROTEINASES IN WOUND HEALING AND SKIN CANCER. Leif R. Lond t, John Romer j, Thomas H. Bugge 1'2, Ken Brown 3, Chnng-Hyun Lee Sogaard I, Jay Degen z and Keld Dana t IFinsen Laboratory, Rigshospitalet, DK-2100 Copenhagen, Denmark, 2Childrens Hospital Research Foundation, Cincinatti, Ohio, USA, JBeatson Institute, Glasgow, United Kingdom Prnteolytic degradation of the extracellular matrix is a central aspect of normal and pathological tissue remodeling events in the skin. By in situ hybridization we find expression of both uPA and uPAR mRNA in the invading cancer cells in human squamons cell skin carcinomas, and during mouse skin wound healing mRNA for uPA and uPAR is expressed by the migrating kemtinocytes, suggesting that the skin cancer cells are imitating these keratinocytes. Following treatment of mouse skin with tumor promoting phorbol esters, a fast induction ofuPA mRNA is seen both in the dermis and epidermis, supporting the notion for an active role of the PAsystem during skin tumor promotion. During skin carcinogenesis induced by DMBA/PMA treatment or by transgane expression of the Rns oncogene under control of the Keratin 5 promoter, only uPA is expressed by papillomas, while both uPA and uPAR are expressed after the transition from papillomas to carcinoma. To investigate the role of plasminogen activation in skin tumor formation, skin tumors were induced in plasminogen-deficient mice by breeding into the transgene Ras expressing mice. Preliminary results show no significant difference between Rasexpressing Pig(4") mice and Pig(+/*) littermate control mice in the type, rate and frequency of skin tumor development. However, the carcinomas showed an abnormal histology with increased extracellular matrix deposition and cell death in the plasminogen-deficient mice compared to the litter mate controls. We have previously reported that skin wound healing is delayed in plasminogen-deficient mice, but also that the wounds eventually will heal. By in situ hybridization we find an expression pattern of the matrix metalloprotcinases gelatinasc B, collagenase-3 and stromeiysin-1 in keratinecytes at the leading edge of epithelial outgrowths similar to that ofuPA and uPAR. These findings suggest that other proteinases are able to substitute for the plasminogen activation system, thereby providing a functional overlap.Thns we have started breeding mice deficient for either gelatinase B or stromelysin-I with plasminogen deficient mice in order to elucidate potential functional overlaps during skin wound healing and skin carcinogenesis.
INHIBITION OF IN VIVO TUMOR GROWTH BY MURINE AND HUMAN UROKINASE RECEPTOR ANTAGONISTS.
86 R E D U C T I O N OF T U M O R C E L L M I G R A T I O N A N D M E T A S T A S I S BY P L A S M I N O G E N A C T I V A T O R INHIBITOR GENE T R A N S F E R M. Prans, K. Wanteriekx, D. Collen and R. D. Gerard Center for Transgene Technology and Gene Therapy, Vlaams lnteruniversitair lnstituut voor Biotechnologie, KU Leuven, Herestraat 49, B-3000 Leuven, Belgie. Urokinase (u-PA), its cellular receptor (u-PAR) and other components of the fibfinolytic cascade have been implicated in various biological processes including tissue remodeling, tumor growth and metastasis. There is a direct correlation between the invasiveness of tumor cells and the level of expression of u-PA, u-PAR and plasminogen activator inhibitor 1 (PAL1). Adanoviral gene transfer has been shown to be a powerful tool for the expression of foreign genes in somatic cells. The aim of this study was to use adenoviral gene transfer of components of the fibrinolytic system to modulate the migratory behavior of human HT-1080 fibrosarcoma/n vitro and the metastatic behavior of HT-1080 ceils in vivo by gene transfer of PAI- 1 and PAI-2. In an in vitro assay using Transwell inserts coated with Matrigel, there was a positive correlation (r= 0.83) between u-PA secretion and tumor cell migration. Migration could be augmented 50% (p=0.027) by adenovirus-mediatud overexpression of u-PA and reduced 30% (p--0.027) by overexpression of PAI-1. Tumor metastasis was studied in vivo in nude mice using two routes of gene transfer. In the first, HT-1080 cells stably expressing luciferase were directly infected with recombinant adenoviruses expressing PAI-1, PAI-2 or control virus and then implanted subcutaneously. Metastasis was quantified at 26 days by assaying luciferase in organ homogenates. Overexpression of PAI-1 and PAI-2 in the tumor cells reduced the percentage of animals with detectable metastasis to the lung by 50% (p-=0.0098) and reduced the burden of metastasis 4-fold (p-~.004 for PAI-1 and p=0.003 for PAI-2). Both tumor incidence and tumor burden in other organs were not significantly affected by PAI gene transfer. In the second, uninfected tumor cells were implanted and liver gene transfer for systemic overexpression of PAI-1 and PAI-2 was performed by tall vein injection of 5x10 8 infectious units of adenovirus. In contrast to the direct infection experiments, overexpressinn of PAI-1 or PAI-2 in the circulation had no significant effect on the incidence of metastasis. PAI-1 failed to achieve significant reduction of the metastatic burden to any organ. However, PAI-2 gene transfer reduced the tumor burden in the brain and lung 6-fold (p<0.01). These results suggest that u-PA activation of plasminogen is involved in the metastasis of human fibrosarcoma cells, that adenoviral gene transfer can modulate both tumor cell migration and metastasis and that the site of expression is critical for the biological activity of PAI-1.
Steven Resenberg, Laura V. Doyle, Robert J. Drummond, Nathalie Dubois-Stringfellow, Hye Yeeng Min, Patriee Pitut, Robert Tressler, Jennifer R. Strattnn-Thomas, and Catherine Zandnnella, Chiron Corporation, 4560 Hortnn Street, Emeryvine, CA 94608. The urokinase: urokinase receptor system has been implicated in tumor growth, angiogenesis, and metastasis, and further has been correlated with progression of a variety of human tumors. However, the lack of a significant phenotype in the uPAR deficient mice has led to significant skepticism on the role of uPAR in in vivo processes. We have expressed and purified antagonists of the routine and human receptors from insect or yeast cells (Min et al., Cancer Res. 56,2428-2433, 1996; Stratton-Thomas etal.,Prot.Eng. 8,463-470, 1995). These antagonists consist of the EGFlike domain of the urokinases, or the domains fused to the constant region of human IgG1. The effects of these molecules on tumor has been tested in 2 models in immnnocomprisad mice: M24Met, human melanoma, and MDA-MB-231, mammary carcinoma, which express human uPAR in vitj..~o, with the highest level found on M24Mct. Dramatic reductions in tumor growth in SCID mice were observed for the M24Met cell line after treatment with hl-48Ig, despite a lack of effeet on the growth of the cell line in vitro. However, in SCID mice lacking complement and natural killer cells, no effect was observed, suggesting the hl-481g was acting as a marker for complement/NK mediated tumor cell killing. In contrast, MDA-MB231 was significantly inhibited by hl-48, and by murine 1-48Ig in SCID/C/NK deficient mice. These results support a previous report .in which expression of the PA system on both tumor cells and stromal cells has been observed for the MDA-MB-231 system (Romer et al., Int. J. Cancer 57, 553-560, 1994), and show that the PA system plays a functional role on both tumor cells and stromal cells in facilitating tumor growth in this in vivo model.
25