While the 72 kDa type IV collagenase (MMP-2) has been broadly implicated in cancer metastasis, the mechanism of activation of the latent zymogen remains unresolved. Highly invasive and metastatic vimentin (VIM)-positive human breast cancer (HBC) cell lines activate MMP-2 when cultured on vitrogen gels, while the poorly-invasive, VIM-negative HBC cell lines do not. Thus, MMP-2 activation may be a rate limiting step in HBC progression. We will extend our analysis to other breast cancer HBC model systems, and to freshly excised HBC cells in short term cultures and xenografted in nude mice to further characterize the significance of MMP-2-activation in vivo (Aim 1). Cellular activation of MMP-2 is induced by culture on interstitial collagen (vitrogen) gels in a cycloheximide-inhibitable manner, and the activator (MMP-2-act) can be extracted from induced cells in the hydrophobic phase of the phase-separable detergent TX114. Activation can be followed using recombinant human MMP2 expressed in MCF-7 HBC cells.
The second aim i s to identify, characterize, purify the MMP-2-activating molecule(s) from the TX114 extracts, and clone the molecule with Dr. Chen. This will facilitate further study of its expression and regulation in relation to HBC progression. Biochemical characterization will facilitate conventional cloning. We will also attempt to identify distinct biofunctional attribute(s) specific for the expression of MMP-2 activation to enable expression cloning of the activator. In the third aim, we wish to extend our preliminary observations of interactions between activated T-lymphocytes and metastatic mouse mammary tumor cells with respect to MMP-2-activation, matrix degradation and basement membrane invasion. The effects of T-cells on metastasis in vivo will be tested in the syngeneic mouse model, and interactions between HBC cell lines and activated T-cells will be examine in vitro and in vivo in nude mice. We will also examine interactions between freshly explanted HBC cells and autologous T-cells. Tumor infiltrating lymphocytes (TILs) will also be isolated, expanded, and examined with respect to their effects on MMP-2 activation, matrix degradation, and tumor cell invasion in both the rodent and human systems. These studies aim to provide a new diagnostic, prognostic, and therapeutic target in the identity of the MMP-2 activation, a molecule which appears to be instrumental in the malignant progression of HBC. We will determine whether and how tumor-activated T-lymphocytes influence MMP-2 activation and/or activity, matrix degradation, and breast cancer cell invasion and metastasis.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA061344-03
Application #
2102083
Study Section
Special Emphasis Panel (SRC (56))
Project Start
1993-07-01
Project End
1997-04-30
Budget Start
1995-05-01
Budget End
1996-04-30
Support Year
3
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Georgetown University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
049515844
City
Washington
State
DC
Country
United States
Zip Code
20057
Thompson, E W; Sung, V; Lavigne, M et al. (1999) LCC15-MB: a vimentin-positive human breast cancer cell line from a femoral bone metastasis. Clin Exp Metastasis 17:193-204
Gilles, C; Bassuk, J A; Pulyaeva, H et al. (1998) SPARC/osteonectin induces matrix metalloproteinase 2 activation in human breast cancer cell lines. Cancer Res 58:5529-36
Sung, V; Gilles, C; Murray, A et al. (1998) The LCC15-MB human breast cancer cell line expresses osteopontin and exhibits an invasive and metastatic phenotype. Exp Cell Res 241:273-84
Sung, V; Cattell, D A; Bueno, J M et al. (1997) Human breast cancer cell metastasis to long bone and soft organs of nude mice: a quantitative assay. Clin Exp Metastasis 15:173-83
Gilles, C; Polette, M; Seiki, M et al. (1997) Implication of collagen type I-induced membrane-type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in the metastatic progression of breast carcinoma. Lab Invest 76:651-60
Yu, M; Sato, H; Seiki, M et al. (1997) Calcium influx inhibits MT1-MMP processing and blocks MMP-2 activation. FEBS Lett 412:568-72
Yu, M; Bowden, E T; Sitlani, J et al. (1997) Tyrosine phosphorylation mediates ConA-induced membrane type 1-matrix metalloproteinase expression and matrix metalloproteinase-2 activation in MDA-MB-231 human breast carcinoma cells. Cancer Res 57:5028-32
Gilles, C; Polette, M; Birembaut, P et al. (1997) Expression of c-ets-1 mRNA is associated with an invasive, EMT-derived phenotype in breast carcinoma cell lines. Clin Exp Metastasis 15:519-26
Pulyaeva, H; Bueno, J; Polette, M et al. (1997) MT1-MMP correlates with MMP-2 activation potential seen after epithelial to mesenchymal transition in human breast carcinoma cells. Clin Exp Metastasis 15:111-20
Yu, M; Sato, H; Seiki, M et al. (1995) Complex regulation of membrane-type matrix metalloproteinase expression and matrix metalloproteinase-2 activation by concanavalin A in MDA-MB-231 human breast cancer cells. Cancer Res 55:3272-7

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