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    Research Grants Awarded

    Targeting Host And Tumoral Mmp-13 In Primary Breast Cancer And Bone Metastasis.

    Grant Mechanism:
    Postdoctoral Fellowships

    Scientific Abstract:
    Background: Matrix metalloproteinases (MMPs) have emerged as important modifiers of key regulatory molecules such as extracellular matrix, cytokines, growth factors and their receptors. Many studies, including our own, have shown that inhibition of all MMPs dramatically reduces primary tumor growth and bone metastasis of breast cancer (BC) cells. The inability to target specific MMPs is probably one of the main reasons that clinical trials of MMP-inhibition have failed. Late stage experimental tumors are not inhibited by broad-spectrum MMP inhibitors, and certain MMPs actually help the host to defend against the tumor growth, while others may fuel the tumor. For this reason, certain MMPs have become known as ?anti-targets? to be avoided, emphasizing the need for specific MMP inhibitors. Another important issue for MMP inhibition is the general toxicity seen so far with most if not all inhibitors tested: Musculo Skeletal Syndrome (MSS). MSS manifests as athralgia and bursitis of the tendon, often causing ?frozen shoulder?. Patients suffering MSS would usually receive a ?drug holiday? and return to the study once the symptoms had subsided, complicating the trial outcomes. For this reason too, specific MMP inhibitors which do not show MSS will be important in translating these important effectors into clinically relevant targets. Hence, we propose that MMP-13 is an important specific MMP target. We have devoted considerable effort into identifying the specific MMPs which manifest in experimental breast cancers, and which compartment they come from (host / stromal or tumoral). In examining 4 human BC cell types growing as orthotopic xenografts in immune-deficient mice, we found a substantial increase (~100 ? 1000 x) in mouse MMP-13 in the mammary fat pad (mfp) xenograft, and to a lesser extent (~10x) in bone metastases, compared to the mfp and bone respectively. The 4T1.2 syngeneic mouse mammary cancer system also shows increased MMP-13 with metastatic capacity, and dramatic induction of stromal MMP-13 in vivo. Preliminary studies with a novel and highly selective MMP-13 inhibitor (MMP-13inh, Pfizer Global) showed inhibition of MDA-MB-231 xenograft growth at the primary site and also in the bone. MMP-13-deficient mice have been acquired and a strain-matched model of metastatic BC has been sourced collaboratively, such that the effects of abrogating tumoral and stromal MMP-13 can be tested independently and together. Pilot experiments verified the targetablity of MMP-13 by co-transfecting pCEP4-MMP-13 and three different shRNA hairpins assembled into pSilencer in human breast cancer cells. Hypothesis, Aims & Research Design: We hypothesise that host/stromal (normal cells surrounding tumor) and/or tumor-derived MMP-13 is critical for the growth, local invasion and/or metastatic spread of breast cancer, and that MMP-13 is an important individual MMP target. Aim 1: To determine the relative contributions of tumoral and host/stromal MMP-13 to breast cancer growth and metastasis using shRNA technology and MMP-13-deficient mice, respectively. We will use shRNA technology to assess the contribution of tumoral MMP-13 on growth and metastasis of both murine and human mammary cancer cells. We will also examine an MMTV-PyMT-Id1 / FVB/n syngeneic model in MMP-13-deficient versus MMP13-intact mouse hosts, to determine the contribution of stromal MMP-13 to the growth and spread of these cells. This model will allow us to independently abrogate the tumoral MMP-13, the host/stromal MMP-13, or both. Aim 2: To perform intervention studies with a novel MMP-13-selective small molecule inhibitor in xenograft and syngeneic models of primary and metastatic breast cancer. We have shown that MMP-13inh is active in prevention mode, and need to determine whether it will halt or regress established disease, both in the primary site and also in the bone environment. Also, to ensure that MMP-13inh is not selectively active in immunocompromised mice, we will extend the study into the immmunocompetent FVB/n mouse mammary tumor model which metastasizes spontaneously to the lungs and lymph node when inoculated into the mammary fat pad. Preliminary data shows considerable expression of MMP13 in MMTV-PyMT-Id1 tumors, consistent with a major host contribution this model. Outcomes & Significance: We will determine and distinguish the contribution of host and tumoral MMP-13 in BC growth and progression using human and mouse BC cell models, and also determine the cellular source of stromal MMP-13. This will allow us to determine the extent to which MMP-13 should be targeted, and will assist in the knowledgeable use of MMP13 inhibitors. Collectively these studies will guide the design and implementation of more effective MMP inhibitors as therapeutics for BC. MMP inhibition continues to hold out hope as a cancer therapy, which we wish to realize.

    Lay Abstract:
    Background: Matrix metalloproteinses (MMPs), a class of enzymes, have been recognized as potential therapeutic targets as they modify many proteins. This can be as part of tissue destruction during invasive migration, but also can be at the molecular level, whereby MMPs can modify different classes of factors such as growth factors, cytokines, and receptors for both. The MMP action can be either enabling or destructive, such that molecular pathways can either depend on MMPs for function, or be abrogated by MMP activity. Inhibitors of these pathways can also be either activated or destroyed by MMPs generating a large range of possibilities. Furthermore, proteases such as MMPs exist in an extensive, interactive system of different protease classes and inhibitors (called the ?protease web?), thus the consequences of MMP action are very complex. Perhaps not surprisingly then, the clinical trials to date using broad spectrum or even somewhat selective MMP inhibitors have not been successful. Certainly a number of MMPs have now been described as ?anti-targets? since they help the host control the tumors, and should not be inhibited. It has become clear that targeting specific MMPs may be required, and we are committed to this approach. In addition to avoiding anti-targets, this approach has the possibility of avoiding a common toxicity that was evident in almost all MMP clinical trials ? Musculo skeletal syndrome (MSS). MSS manifests as athralgia and bursitis of the tendon, often causing ?frozen shoulder?. Patients suffering MSS would usually receive a ?drug holiday? and return to the study once the symptoms had subsided. Since surrogate markers for efficacy at the tumor were not available for clinical trial, the possibility remains that individual were removed from therapy only when the levels became efficacious, which may also have contributed to the poor trial outcomes. We have performed experiments to determine the most likely candidates to be targeted and MMP-13 was found to be dramatically increased in the normal cells/ tissue surrounding the cancer cells in series of experimental breast cancers. Also others have examined MMP-13 expression in patient material, where it corresponded to loss of the outside structure that usually contains the tumor. We also found that the level of MMP-13 made by the more aggressive human breast cancer cells was increased when grown in the mouse. Recently, we obtained very promising results with a small molecule inhibitor which is highly selective for MMP-13 inhibition developed by Pfizer Global. This inhibitor reduces breast cancer growth at the primary site and slowed the progress of osteolytic bone lesions, supporting an involvement of MMP-13 in breast cancer growth at primary site and bone damage. We thus hypothesize that both tumor-derived and host/stromal (normal cells surrounding tumor) MMP-13 may be important for growth and spread of breast cancer. We will determine the role of MMP-13 made by the cancer cells and stroma, respectively by extending the inhibitor work and using the MMP-13-deficient versus MMP-13-intact mice. The study has the potential for translation into clinical trial should it succeed. Objective/Hypothesis: We aim to use novel approaches to target, separately, the MMP-13 produced by the normal cells surrounding host tissue and the tumor cells, and determine effects on breast tumor growth and spread. Specific Aims: We aim to study the role of mouse (host/stromal) and tumoral (human) MMP-13 has in breast cancer, and examine further the novel MMP-13-specific inhibitor. Study Design: In targeting the tumoral MMP-13, we will use a method called short-interfering RNA which potently reduces the specific protein. This will be done both with human xenografted breast cancer cells, as well as syngeneic mouse mammary cancer cells. Further, use of MMP-13-intact mice versus MMP-13-deficient mice will determine the role of stromal MMP-13 produced by host / stromal cells in breast cancer growth and bone damage. Potential Outcomes and Benefits of the Research: This study will allow us to determine the extent to which MMP-13 should be targeted, and will assist in the knowledgeable use of such inhibitors. The pre-clinical screening of MMP-13 inhibitor will offer the exciting prospect of new cancer therapeutics. Being a late-stage manifestation, bone metastasis may represent an important clinical trial setting for MMP inhibitors.