Extracellular Matrix Pathology staff

Mission Statement of the Extracellular Matrix Pathology Section:

The research goal of the Extracellular Matrix Pathology Section, LP, CCR, NCI is to further our understanding of the mechanisms of cell-extracellular matrix interactions which regulate cell behavior during tumor progression and angiogenesis. In particular we are interested in the regulation of cell invasion and cell growth. These processes require remodeling of the extracellular matrix that occurs in a spatially and temporally controlled fashion. This involves both proteases and protease inhibitor activities that are tightly regulated.

The tissue inhibitor of metalloproteinase (TIMP) family consists of four members: TIMP-1, TIMP-2, TIMP-3 and TIMP-4. All TIMP family members are secreted into the extracellular milieu and have a similar size core protein of 182-194 amino acids with 12 cysteine residues that form six intramolecular disulfides. Correct folding of the peptide backbone and formation of these disulfide bonds are required for functional MMP inhibitor activity. MMP inhibitor activity is further localized to the three amino-terminal disulfide loops and requires a free amino terminal cysteine residue. Studies have shown that the transcription of individual TIMP genes are regulated independently of one another.

TIMPs inhibit cell invasion (both tumor and endothelial cells) through reconstituted basement membranes in vitro, principally via inhibition of metalloproteinase activity. Both TIMP-1 and TIMP-2 demonstrate cell growth regulating and erythroid potentiating activities (EPA) that appear to be functionally distinct from inhibition of proteases. The specific aims of these studies are: 1) to understand the relationship of protease inhibitor activity to the growth regulating activity of TIMPs; 2) to define the mechanisms through which individual members of the TIMP family regulate cell growth. To accomplish these goals we have developed a series of modified TIMP proteins and expression vectors, as well as both in vitro and in vivo models to examine the effects of TIMPs on cell growth. Using these reagents and model systems we have demonstrated that modulation of endothelial and tumor cell growth by TIMPs in vitro and in vivo is independent of the protease inhibitor activity of these proteins. In addition, direct cell surface binding of TIMPs has been demonstrated suggesting the presence of putative cell surface receptors for these extracellular matrix protease inhibitors. Signal transduction studies suggest that TIMP binding activates adenylate cyclase, protein kinase A and the protein tyrosine phosphatase SHP-1. Further experiments are directed at:

1) identification and characterization of TIMP receptors; 2) identification of specific peptide sequences responsible for the cell growth modulating properties of TIMPs; 3) proteomic and cDNA micro-array characterization of cellular responses to TIMPs both in vitro and in vivo. Our findings confirm the anti-angiogenic activity of TIMPs, as well as a unique growth inhibitory activity of TIMP-2 on human microvascular endothelial cells. Identification and characterization of TIMP-2-derived peptide sequences, and/or TIMP receptors may lead to development of novel anticancer therapies.

Recent Research Findings

1. Compared isogenic human colorectal cancer cell lines by in vitro morphology, xenograft morphology tumorigenicity, mitotic an apoptotic indices, FACS analysis of TNF_ receptor expression and anti-FAS induction of apoptosis, as well as in vitro adhesion and migration. Our findings suggest that comparison of the SW480 and SW 620 cells may serve as an interesting model for examining the invasive/metastatic phenotype (J. Pathol. 192:446-454, 2000).
   

2. Compared isogenic human colorectal cancer cell lines, primary (SW480) versus metastatic (SW620), by differential display. Demonstrated decreased expression of the CC3 tumor suppressor gene and tissue inhibitor of metalloproteinase-3 (TIMP-3) gene in the metastatic cell line (SW620). These findings were confirmed by Northern blot analysis. In addition we demonstrate that TIMP-1 expression is markedly increased in the metastatic cell line (SW620) (J. Pathol. 192: 455-459, 2000).

3. Demonstrated the molecular mechanism of TIMP-2 growth inhibition of EGF-stimulated human lung adenocarcinoma cells. Discovered that TIMP-2 mediated inhibition of tyrosine kinase growth factor stimulation is not dependent on inhibition of metalloproteinase activity, but is the result of adenylate cyclase activation and subsequent selective induction of SH2 protein tyrosine phosphatase, SHP-1, activity and association with the EGFR (J. Biol. Chem. 276: 3203-3214, 2001).
   

4. Demonstrated that TIMP-1 alters the in vivo growth of Burkitt¹s lymphoma by divergent effects on tumor growth and angiogenesis. It is well established that growth of human Burkitt¹s lymphoma xenografts in the nude mouse model results initially in rapid tumor growth followed by spontaneous necrosis and complete regression. Investigators have unsuccessfully attempted to identify the molecular mechanisms responsible for the regression of these tumors. We demonstrate that forced expression of TIMP-1 in the EBV-negative JD38 Burkitt¹s lymphoma cell line results in an initially enhancement of tumor growth consistent with the anti-apoptotic effect previously described in vitro. These tumors subsequently undergo central necrosis and complete regression. The molecular mechanism of this effect is demonstrated as TIMP-1 inhibition of tumor-assoicated angiogenesis (Am. J. Pathol. 158: 1207-1215, 2001).
   
   

5. Defined the molecular mechanism associated with reduction in tumorigenicity of EGFR antisense treatment of human ovarian carcinoma cells. We have characterized the altered cellular morphology, attachment, integrin expression, protease/inhibitor profile and migration of NIH:OVCAR-8 cells transfected with antisense EGFR. We demonstrate that altered morphology and decreased laminin-1 adhesion, as well as a selective decrease in MMP-9 activity. Alteration in laminin-1 attachment is mediated by selective decrease in expression of the integrin _6 subunit, but not _3 subunit, in the EGFR antisense cells. These findings suggest that decreases in MMP-9 and _6 subunit could be used as clinical endpoints in anti-EGFR therapies in human ovarian carcinoma (JNCI, In press, 2001.)

New and ongoing initiatives

1. Development of a novel in vivo angiogenesis assay. During the past year we have refined our novel in vivo angiogenesis assay and demonstrated the reproducibility of this system from assay to assay. This assay system allows the rapid (~10 days) assessment of activity for angiogenic factors or angiogenesis inhibitors in vivo. The results are quantitative and highly reproducible.
   

2. We have characterized the in vitro and in vivo anti-angiogenic activities of TIMP-2 and demonstrated that these effects are dissociable from MMP inhibitor activity. This suggest that identification of the TIMP-2 active fragment could lead to the development of novel angiogenesis inhibitors that would complement the MMP inhibitors already in clinical testing. 3. We have further characterized the molecular mechanism of TIMP-1 and TIMP-2 effects on cell growth with identification of domain sequences responsible and construction of loss/gain of function mutants. These experiments will aid in further refinement of our understanding of TIMP biology.

Recent Publications

1. Galateau-Salle, F. B., Luna, R. E., Horiba, K., Sheppard, M. N., Hayashi, T., Fleming, M. V., Colby, T. V., Bennett, W., Harris, C. C., Stetler-Stevenson, W. G., Liotta, L., Ferrans, V. and J.Travis, W. D.:
   Matrix metalloproteinases and tissue inhibitors of metalloproteinases in bronchial squamous preinvasive lesions. Human Pathol. 31: 296-305, 2000.

2. Elliot, S. J., Striker, L. J., Connor, E., Stetler-Stevenson, W.G., W. C., Blagg, C. R. and Striker, G. E.: Pentosan polysulfate decreases proliferation and extracellular matrix deposition by vascular smooth muscle cells isolated from failed hemodialysis access grafts. Clin. Nephrol. 54:121-127, 2000.

3. Lenz, O., Elliot, S. J. and Stetler-Stevenson, W. G.: Matrix metalloproteinases in renal development and disease. J. Amer. Soc. Nephrol. 11: 574-581, 2000.

4. Taraboletti, G., Sonzogni, L., Vergani, V., Hosseini, G., Ceruti, R., Ghilardi, C., Bastone, A.,, Toschi, E., Borsotti, P., Scanziani, E., Giavazzi, R., Pepper, M. S., Stetler-Stevenson, W. G. and Bani, M. R.:
   Posttranscriptional stimulation of endothelial cell matrix metalloproteinases 2 and 1 by endothelioma cells. Exp. Cell Res. 258:
   384-394, 2000.

5. Hewitt, R.E., Brown, K.E., Corcoran, M. and Stetler-Stevenson,
   W.G.: Increased expression of tissue inhibitor of metalloproteinases type 1 (TIMP-1) in a more tumourigenic colon cancer cell line. J. Pathol. 192(4):455-459, 2000.

6. Hewitt, R.E., McMarlin, A., Kleiner, D., Wersto, R., Martin, P.,
   Tsokos, M., Stamp, G.W., Stetler-Stevenson, W.G.: Validation of a model of colon cancer progression. J. Pathol, 192(4):446-454, 2000.

7. Birkedal-Hansen, B., Pavelic, Z.P., Gluckman, J. L., Stambrook, P.,
   Li, Y.-Q. and Stetler-Stevenson, W. G.: MMP and TIMP Gene Expression in Head and Neck Squamous Cell Carcinoma and Adjacent Tissues. Oral Diseases: 6:
   376-382, 2000.

8. Holten-Andersen, M. N., Stephens, R. W., Nielsen, H. J., Murphy G.,
   Christensen, I. J., Stetler-Stevenson, W. and Brünner, N. : High Preoperative Plasma Tissue Inhibitor of Metalloproteinase-1 Levels Are Associated with Short Survival of Patients with Colorectal Cancer. Clin Cancer Res. 6: 4292-4299, 2000.

9. Hoegy, S.E., Oh, H.-R., Corcoran, M.L., and Stetler-Stevenson, W. G.:
   Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) Suppresses TKR-Growth Factor Signaling Independnent of Metalloproteinase Inhibition. J. Biol. Chem. 276: 3203-3214, 2001.

10. Guedez, L., Mansoor, A., Birkedal-Hansen, B., Lim, M.S., Fukushima,
    P., Venzon, D., Stetler-Stevenson, W. G. and Stetler-Stevenson, M.: TIMP-1 Regulation of IL-10 in B Cell Differentiation and Lymphomagenesis. Blood:
    97: 1796-1802, 2001.

11. Guedez, L., McMarlin, A. J., Kingma, D. W., Bennett, T. A.,
    Stetler-Stevenson, M. and Stetler-Stevenson, W. G.: Tissue Inhibitor of Metalloproteinases (TIMP)-1 Alters The Tumorigenicity of Butkitt¹s Lymphoma Via Both Growth Promoting and Dominant Anti-Angiogenic Effects. Amer. J. Pathol. 158: 1207-1215, 2001.

12. Vergani, V., Garofalo, A., Bani, M.R., Borsotti, P., Parker, M.P.,
    Drudis, T., Mazzarol, G., Viale, G., Giavazzi, R., Stetler-Stevenson, W.G. and Taraboletti, G.: Inhibition of matrix metalloproteinases by over-expression of tissue inhibitor of metalloproteinase-2 inhibits the growth of experimental hemangiomas. Int J Cancer. 91(2): 241-247, 2001.

13. Alpler, Ö., Bergmann-Leitner, S., Bennett, T. Hacker, N.F., Stromberg,
    K. and Stetler-Stevenson, W. G.: EGFR-Antisense In Ovarian Carcinoma Cells Reduces Integrin and Metalloproteinase-Mediated Attachment/Migration. Journal Natl. Cancer Inst., 93: 1375-1384, 2001.

14. Seftor R.E.B., Seftor, E. A., Meltzer, P. S., Gardner, L.M.G., Biban,
    M., Koshikawa, N., Schateemen, G. C., Stetler-Stevenson, W. G., Quaranta, V. and Hendrix, M.J.C. Cooperative Interactions of Laminin __2, MMP-2 and MT-1-MMP are Required for Mimicry of Embryonic Vasculogenesis by Aggressive Melanoma, submitted.

15. Shankavaram, U. T., Lai, W.-C., Netzel-Arnett, S., Mangan, P. R.,
    Ardans, J.A., Caterina, N., Stetler-Stevenson, W.G., Birkedal-Hansen, H. and Wahl, L.M: Monocyte Membrane Type I-Matrix Metalloproteinase:
    Prostaglandin-Dependent Regulation and Role in MMP-2 Activation. J. Biol.
    Chem., In press, 2001.

16. Stetler-Stevenson, W. G. and Yu, A.: Proteases in invasion: matrix metalloproteinases. Sem. Cancer Biol. 11: 143-153, 2001.

17. Stetler-Stevenson, W. G. and Kleiner, D. E., Jr.: Molecular Biology of
    Cancer: Molecular Mechanisms of Cancer Invasion and Metastasis in Cancer Principles and Practice of Oncology, DeVita, V., Hellman, S. and Rosenberg, S., eds. Lippincot, William & Wilkins, Philadelphia, 2001, pp. 123-136.

18. Bennett, T. A. and Stetler-Stevenson, W. G.: Matrix
    Metalloproteinases (Matrixins) and Their Inhibitors (TIMPS) in Angiogenesis. In Tumor Angiogenesis and Microcirulation, Voest, E. E. and D¹Amore, P.A., eds. Marcel Dekker, Inc., New York, 2001, pp. 29-57.

19. Stetler-Stevenson, W. G.: The Role of Matrix Metalloproteinases in Tumor
    Invasion, Metastasis and Angiogenesis. Surg. Clin. N. Amer. 10: 383-392, 2001.