Signe Ingvarsen:
Interfering with membrane type matrix metalloproteases in processes of extracellular matrix turnover

Date: 16-01-2012    Supervisor: Karen Skriver

Matrix metalloproteases (MMPs) are centrally engaged in the processes of extracellular matrix (ECM) turnover that occur both during normal development and in connection with cancer growth, invasion and metastasis. The work presented in this thesis is focused on two membrane anchored MMPs, MT1‐and MT2‐ MMP. MT1‐MMP is a collagenolytic enzyme which plays an important role in pericellular ECM remodeling and the protease is critically involved during several stages of tumor progression. In addition to its ability to directly cleave collagens and other ECM proteins, MT1‐MMP also contributes to ECM degradation through the activation of proMMP‐2, the proform of the secreted gelatinase MMP‐2. In contrast to the wellestablished role of MT1‐MMP in ECM remodeling, much less is known about the function of MT2‐MMP, but the enzyme displays activity against different ECM proteins, including collagens, and has also been linked to tumorigenesis. In order to enable studies on MT1‐and MT2‐MMP function in vitro and in vivo, the aim of this PhD study was to develop monoclonal antibodies (mAbs) which interfere with the function of these proteases. The thesis is divided into two parts, a major part concerning MT1‐MMP and a minor part concerning MT2‐MMP.

The first part describes the generation of two panels of anti‐MT1‐MMP mAbs by immunization of MT1‐ MMP deficient mice with recombinant murine MT1‐MMP. From each antibody panel a mAb interfering with MT1‐MMP function was identified and characterized in greater detail. mAb‐1 induces cellular proMMP‐2 activation through the formation of MT1‐MMP dimers on the cell surface and was used in a functional study to demonstrate the regulatory role of MT1‐MMP dimerization in the process of proMMP‐2 activation. mAb 9E8 was identified as a selective inhibitor of MT1‐MMP dependent proMMP‐2 activation. While mAb 9E8 completely prevents cellular proMMP‐2 activation it has no effect on the collagenolytic or general proteolytic activity of MT1‐MMP and therefore it provides a unique opportunity to discriminate between the MT1‐MMP effects exerted through proMMP‐2 activation and the effects exerted through direct cleavage of ECM components.

The second part of the thesis describes the expression, purification and characterization of recombinant murine MT2‐MMP protein with the aim of producing protein which can be used as an antigen for the generation of anti‐MT2‐MMP mAbs and for the characterization of MT2‐MMP´s proteolytic activity. Although the progress of this project was hindered by methodological difficulties related to protein expression and purification, initial characterization of the purified protein suggests that the proteolytic activity of MT2‐MMP differs significantly from that of MT1‐MMP and emphasizes the need for further characterization of MT2‐MMP.