Kasper Almholt

CANCER INVASION AND TISSUE REMODELING : COMMON THEMES IN PROTEOLYTIC MATRIX DEGRADATION

Analysis of extracellular matrix degradation systems has led to the insight that in cancer invasion there is often crucial interplay between cancer cells and several types of surrounding non-neoplastic stromal cells. Likewise, in normal tissue remodeling processes, the synthesis of proteolytic components is often distributed between several cell types, and there are strong similarities between neoplastic and non-neoplastic processes in the same tissue. Thus, tissue remodeling events are excellent models for studies of extracellular proteolysis in cancer. This has become even clearer by recent analyses of genetically modified mice.

Tumor Cell–Derived and Macrophage-Derived Cathepsin B Promotes Progression and Lung Metastasis of Mammary Cancer

Proteolysis in close vicinity of tumor cells is a hallmark of cancer invasion and metastasis. We show here that mouse mammary tumor virus-polyoma middle T antigen (PyMT) transgenic mice deficient for the cysteine protease cathepsin B (CTSB) exhibited a significantly delayed onset and reduced growth rate of mammary cancers compared with wild-type PyMT mice. Lung metastasis volumes were significantly reduced in PyMT;ctsb(+/-), an effect that was not further enhanced in PyMT;ctsb(-/-) mice. Furthermore, lung colonization studies of PyMT cells with different CTSB genotypes injected into congenic wild-type mice and in vitro Matrigel invasion assays confirmed a specific role for tumor-derived CTSB in invasion and metastasis. Interestingly, cell surface labeling of cysteine cathepsins by the active site probe DCG-04 detected up-regulation of cathepsin X on PyMT;ctsb(-/-) cells. Treatment of cells with a neutralizing anti-cathepsin X antibody significantly reduced Matrigel invasion of PyMT;ctsb(-/-) cells but did not affect invasion of PyMT;ctsb(+/+) or PyMT;ctsb(+/-) cells, indicating a compensatory function of cathepsin X in CTSB-deficient tumor cells. Finally, an adoptive transfer model, in which ctsb(+/+), ctsb(+/-), and ctsb(-/-) recipient mice were challenged with PyMT;ctsb(+/+) cells, was used to address the role of stroma-derived CTSB in lung metastasis formation. Notably, ctsb(-/-) mice showed reduced number and volume of lung colonies, and infiltrating macrophages showed a strongly up-regulated expression of CTSB within metastatic cell populations. These results indicate that both cancer cell-derived and stroma cell-derived (i.e., macrophages) CTSB plays an important role in tumor progression and metastasis.

Reduced metastasis of transgenic mammary cancer in urokinase‐deficient mice

A prominent phenotype of plasmin deficiency in mice is reduced metastasis in the MMTV‐PymT transgenic breast cancer model. Proteolytically active plasmin is generated from inactive plasminogen by one of 2 activators, uPA or tPA. We now find that uPA deficiency alone significantly reduces metastasis >7‐fold in the MMTV‐PymT model. We studied a cohort of 55 MMTV‐PymT transgenic mice, either uPA‐deficient or wild‐type controls. Tumor incidence, latency, growth rate and final primary tumor burden were not significantly affected by uPA deficiency. In contrast, average lung metastasis volume was reduced from 1.58 mm3 in wild‐type controls to 0.21 mm3 in uPA‐deficient mice (p = 0.023). Tumor cell dissemination to brachial lymph nodes was also reduced from 53% (28/53) in wild‐type controls to 31% (17/54) in uPA‐deficient mice (p = 0.032). Mice without plasminogen display a severe pleiotropic phenotype. By comparison, spontaneous phenotypes are modest in uPA‐deficient mice, probably because they still have active tPA. We show that metastasis is strongly and selectively decreased in uPA‐deficient mice, suggesting that uPA‐directed antimetastatic therapy would be efficacious and have limited side effects.

Plasminogen activation independent of uPA and tPA maintains wound healing in gene-deficient mice

Simultaneous ablation of the two known activators of plasminogen (Plg), urokinase‐type (uPA) and the tissue‐type (tPA), results in a substantial delay in skin wound healing. However, wound closure and epidermal re‐epithelialization are significantly less impaired in uPA;tPA double‐deficient mice than in Plg‐deficient mice. Skin wounds in uPA;tPA‐deficient mice treated with the broad‐spectrum matrix metalloproteinase (MMP) inhibitor galardin (N‐[(2R)‐2‐(hydroxamido‐carbonylmethyl)‐4‐methylpentanoyl]‐L‐tryptophan methylamide) eventually heal, whereas skin wounds in galardin‐treated Plg‐deficient mice do not heal. Furthermore, plasmin is biochemically detectable in wound extracts from uPA;tPA double‐deficient mice. In vivo administration of a plasma kallikrein (pKal)‐selective form of the serine protease inhibitor ecotin exacerbates the healing impairment of uPA;tPA double‐deficient wounds to a degree indistinguishable from that observed in Plg‐deficient mice, and completely blocks the activity of pKal, but not uPA and tPA in wound extracts. These findings demonstrate that an additional plasminogen activator provides sufficient plasmin activity to sustain the healing process albeit at decreased speed in the absence of uPA, tPA and galardin‐sensitive MMPs and suggest that pKal plays a role in plasmin generation.

Stromal Cell Involvement in Cancer

Solid tumors co-opt the bodys endogenous extracellular proteolytic machinery for their invasion and metastasis. This is supported by a large number of independent observations ranging from histochemical and prognostic studies of cancer patient material to animal experiments. There are several extracellular proteolytic systems that are relevant in the context of cancer, but the plasminogen activation (PA) system and the matrix metalloproteases (MMPs) remain the most thoroughly investigated. Localization studies by immunohistochemistry and in situ mRNA hybridization in tumors of common human cancers have repeatedly identified members of the PA and MMP systems in stromal cells. The cancer cells, of epithelial origin, contribute PA and MMP components in some cases, but their contribution fades in comparison with the overwhelming expression of proteolytic components by fibroblasts, macrophages, endothelial cells, and other stromal cells. Ideal animal models of human cancers should recapitulate this fundamental proteolytic aspect of tumor biology. However, in the transplantable tumor models where PA or MMP components have been studied at the cellular level in vivo, this is most often not the case. Transgenic cancer models may provide a closer parallel to the human situation, in that PA and MMP components are synthesized by the tumor stroma. The pivotal role of stromal cells has been confirmed experimentally in mouse models in which the expression pattern of proteolytic components is strongly reminiscent of human tumors. In these models it is possible to reconstitute the wild-type tumor characteristics of proteolytically deficient tumor-bearing mice by transplantation with wild-type fibroblasts or hemapoietic cells. These studies collectively show that cancer-associated proteolysis is a collaborative effort of malignant cancer cells and various stromal cells--a collaboration in which stromal cells contribute the majority of the active proteolytic components that are necessary for the invasive behavior of the tumors. This cellular division of labor positions the stromal cells as prime targets for future research and possibly therapy. Vascular endothelial cells are already the focus of intense therapeutically relevant research, but tumor-associated fibroblasts, macrophages, neutrophils, lymphendothelial cells, etc. provide additional largely unexplored territory in the ongoing search for efficient countermeasures against invasive cancer.

Metastasis of transgenic breast cancer in plasminogen activator inhibitor-1 gene-deficient mice

The plasminogen activator inhibitor-1 (PAI-1) blocks the activation of plasmin(ogen), an extracellular protease vital to cancer invasion. PAI-1 is like the corresponding plasminogen activator uPA (urokinase-type plasminogen activator) consistently expressed in human breast cancer. Paradoxically, high levels of PAI-1 as well as uPA are equally associated with poor prognosis in cancer patients. PAI-1 is thought to play a vital role for the controlled extracellular proteolysis during tumor neovascularization. We have studied the effect of PAI-1 deficiency in a transgenic mouse model of metastasizing breast cancer. In these tumors, the expression pattern of uPA and PAI-1 resembles that of human ductal breast cancer and plasminogen is required for efficient metastasis. In a cohort of 63 transgenic mice that were either PAI-1-deficient or wild-type sibling controls, primary tumor growth and vascular density were unaffected by PAI-1 status. PAI-1 deficiency also did not significantly affect the lung metastatic burden. These results agree with the virtual lack of spontaneous phenotype in PAI-1-deficient mice and humans and may reflect that the plasminogen activation reaction is not rate limiting for tumor vascularization and metastasis, or that there is a functional redundancy between PAI-1 and other inhibitors of the uPA/plasmin system, masking the effect of PAI-1 deficiency.

Lung Metastasis Fails in MMTV-PyMT Oncomice Lacking S100A4 Due to a T-Cell Deficiency in Primary Tumors

Interactions between tumor and stroma cells are essential for the progression of cancer from its initial growth at a primary site to its metastasis to distant organs. The metastasis-stimulating protein S100A4 exerts its function as a stroma cell-derived factor. Genetic depletion of S100A4 significantly reduced the metastatic burden in lungs of PyMT-induced mammary tumors. In S100A4(+/+) PyMT mice, massive leukocyte infiltration at the site of the growing tumor at the stage of malignant transition was associated with increased concentration of extracellular S100A4 in the tumor microenvironment. In contrast, in S100A4(-/-) PyMT tumors, a significant suppression of T-cell infiltration was documented at the transition period. In vitro, the S100A4 protein mediated the attraction of T cells. Moreover, S100A4(+/+), but not S100A4(-/-), fibroblasts stimulated the invasion of T lymphocytes into fibroblast monolayers. In vivo, the presence of S100A4(+/+), but not S100A4(-/-), fibroblasts significantly stimulated the attraction of T lymphocytes to the site of the growing tumor. Increased levels of T cells were also observed in the premetastatic lungs of tumor-bearing mice primed to metastasize by S100A4(+/+) fibroblasts. Treatment of T cells with the S100A4 protein stimulated production of cytokines, particularly granulocyte colony-stimulating factor and eotaxin-2. The same cytokines were detected in the fluid of S100A4(+/+) PyMT tumors at the transition period. We suggest that release of S100A4 in the primary tumor stimulates infiltration of T cells and activates secretion of cytokines, thus triggering sequential events that fuel tumor cells to metastasize. Similar processes could occur in the premetastatic lungs, facilitating generation of inflammatory milieu favorable for metastasis formation.

Metastasis is strongly reduced by the matrix metalloproteinase inhibitor Galardin in the MMTV-PymT transgenic breast cancer model

Matrix metalloproteinases (MMP) have several roles that influence cancer progression and dissemination. However, low molecular weight metalloproteinase inhibitors (MPI) have not yet been tested in transgenic/spontaneous metastasis models. We have tested Galardin/GM6001, a potent MPI that reacts with most MMPs, in the MMTV-PymT transgenic breast cancer model. We followed a cohort of 81 MMTV-PymT transgenic mice that received Galardin, placebo, or no treatment. Galardin treatment was started at age 6 weeks with 100 mg/kg/d, and all mice were killed at age 13.5 weeks. Galardin treatment significantly reduced primary tumor growth. Final tumor burden in Galardin-treated mice was 1.69 cm3 compared with 3.29 cm3 in placebo-treated mice (t test, P = 0.0014). We quantified the total lung metastasis volume in the same cohort of mice. The median metastasis volume was 0.003 mm3 in Galardin-treated mice compared with 0.56 mm3 in placebo-treated mice (t test, P < 0.0001). Thus, metastasis burden was reduced more than 100-fold, whereas primary tumor size was reduced only 2-fold. We also found that primary tumors from Galardin-treated mice exhibited a lower histopathologic tumor grade, increased collagen deposition, and increased MMP-2 activity. MMPs are known to have tumor-promoting and tumor-inhibitory effects, and several clinical trials of broad-spectrum MPIs have failed to show promising effects. The very potent antimetastatic effect of Galardin in the MMTV-PymT model does, however, show that it may be possible to find broad-spectrum MPIs with favorable inhibition profiles, or perhaps combinations of monospecific MPIs, for future clinical application. [Mol Cancer Ther 2008;7(9):2758–67]

Extracellular Proteolysis in Transgenic Mouse Models of Breast Cancer

Growth and invasion of breast cancer require extracellular proteolysis in order to physically restructure the tissue microenvironment of the mammary gland. This pathological tissue remodeling process depends on a collaboration of epithelial and stromal cells. In fact, the majority of extracellular proteases are provided by stromal cells rather than cancer cells. This distinct expression pattern is seen in human breast cancers and also in transgenic mouse models of breast cancer. The similar expression patterns suggest that transgenic mouse models are ideally suited to study the role of extracellular proteases in cancer progression. Here we give a status report on protease intervention studies in transgenic models. These studies demonstrate that proteases are involved in all stages of breast cancer progression from carcinogenesis to metastasis. Transgenic models are now beginning to provide vital mechanistic insight that will allow us to combat breast cancer invasion and metastasis with new protease-targeted drugs.

Antibody-mediated Targeting of the Urokinase-type Plasminogen Activator Proteolytic Function Neutralizes Fibrinolysis in Vivo

Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling processes. Most of our understanding of the role of uPA in vivo is derived from studies using genetargeted uPA-deficient mice. To enable in vivo studies on the specific interference with uPA functionality in mouse models, we have now developed murine monoclonal antibodies (mAbs) directed against murine uPA by immunization of uPA-deficient mice with the recombinant protein. Guided by enzyme-linked immunosorbent assay, Western blotting, surface plasmon resonance, and enzyme kinetic analyses, we have selected two highly potent and inhibitory anti-uPA mAbs (mU1 and mU3). Both mAbs recognize epitopes located on the B-chain of uPA that encompasses the catalytic site. In enzyme activity assays in vitro, mU1 blocked uPA-catalyzed plasminogen activation as well as plasmin-mediated pro-uPA activation, whereas mU3 only was directed against the first of these reactions. We additionally provide evidence that mU1, but not mU3, successfully targets uPA-dependent processes in vivo. Hence, systemic administration of mU1 (i) rescued mice treated with a uPA-activable anthrax protoxin and (ii) impaired uPA-mediated hepatic fibrinolysis in tissue-type plasminogen activator (tPA)-deficient mice, resulting in a phenotype mimicking that of uPA;tPA double deficient mice. Importantly, this is the first report demonstrating specific antagonist-directed targeting of mouse uPA at the enzyme activity level in a normal physiological process in vivo.