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.
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