Boye Schnack Nielsen

Cancer invasion and tissue remodeling : cooperation of protease systems and cell types

Proteolytic degradation of the extracellular matrix plays a crucial role in both cancer invasion and non‐neoplastic tissue remodeling processes. In human cancers the components of matrix degrading protease systems (uPA, uPAR, PAI‐1 and MMPs) can be expressed by either the non‐neoplastic stromal cells, the cancer cells or both. Studies of the prognostic impact of these components in human cancer and the effect of targeted gene inactivation on cancer metastasis in mice support the assumption that proteases promote cancer progression, independent of whether they are expressed by cancer cells or stromal cells. The pattern of expression of components of protease systems is usually very similar in different cases of the same type of cancer, while it varies between different types of cancer. There are intriguing similarities between the cellular expression pattern of components of protease systems seen in cancer invasion and in certain types of non‐neoplastic tissue remodeling. We propose that cancer invasion can be viewed as tissue remodeling gone out of control. The stromal cell involvement in cancer invasion represents a new paradigm with important implications for cancer pathophysiology and cancer therapy.

Functional overlap between two classes of matrix-degrading proteases in wound healing.

Retarded wound healing was found in mice deficient in the serine protease precursor plasminogen, as well as in wild‐type mice treated with the metalloprotease inhibitor galardin, but in both cases wound closure was ultimately completed in all mice within 60 days. The expression of several matrix metalloproteases in keratinocytes migrating to cover the wound was strongly enhanced by galardin treatment. However, when plasminogen‐deficient mice were treated with galardin, healing was completely arrested and wound closure was not seen during an observation period of 100 days, demonstrating that protease activity is essential for skin wound healing. The requirement for both plasminogen deficiency and metalloprotease inhibition for complete inhibition of the healing process indicates that there is a functional overlap between the two classes of matrix‐degrading proteases, probably in the dissection of the fibrin‐rich provisional matrix by migrating keratinocytes. Each class alone is capable of maintaining sufficient keratinocyte migration to regenerate the epidermal surface, although this function would normally be performed by both classes acting in parallel. Since there are strong similarities between the proteolytic mechanisms in wound healing and cancer invasion, these results predict that complete arrest of this latter process in therapeutic settings will require the use of inhibitors of both classes of proteases.

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.

92 kDa type IV collagenase (MMP-9) is expressed in neutrophils and macrophages but not in malignant epithelial cells in human colon cancer

Degradation of the extracellular matrix during cancer invasion is accomplished by the concerted action of several proteolytic enzymes, including matrix metalloproteinases (MMPs). We have studied the immunohistochemical localization of one of these enzymes, 92‐kDa type IV collagenase (MMP‐9), in short‐term fixed specimens of 19 colon adenocarcinomas and 2 biopsies of adjacent normal colon. Staining was confined to neutrophils and macrophages, as identified by double staining. All neutrophils were positive in all cases. Some positively stained tumor‐infiltrating macrophages were seen in 6 (32%) of the tumors, located adjacent to invasive tumor glands. No cancer cells were stained in any of the cases. In normal colon tissue, staining was only seen of scattered neutrophils in vessels and of macrophages in Peyers patches. Routinely processed specimens from 7 of the 19 carcinomas were analyzed by in situ hybridization. In agreement with previous results, a MMP‐9 mRNA signal was in all cases seen in a subpopulation of tissue macrophages surrounding invasive tumor glands, while no MMP‐9 mRNA was detected in any other cell types, including neutrophils and cancer cells. Our results indicate that in this type of cancer all neutrophils contain MMP‐9, which has been produced before they infiltrate the tumors; that a subpopulation of the tumor‐infiltrating macrophages most likely in all cases produces MMP‐9 but that the content of this protein is low due to a rapid turnover and that malignant epithelial cells do not produce or contain detectable amounts of MMP‐9. These findings extend previous results indicating that stromal cells are actively involved in the generation and regulation of extracellular proteolysis during cancer invasion.

Reduced metastasis of Polyoma virus middle T antigen-induced mammary cancer in plasminogen-deficient mice

To investigate the role of plasmin(ogen) in mammary tumor development and progression, plasminogen- deficient mice were crossed with transgenic mice expressing Polyoma middle T antigen under the control of the mouse mammary tumor virus long terminal repeat. Virgin females carrying the Polyoma middle T antigen uniformly developed multiple, bilateral mammary tumors, regardless of the presence or absence of circulating plasminogen. Both the age at which these tumors became palpable and subsequent tumor growth were indistinguishable between plasminogen-deficient mice and plasminogen-expressing littermates. However, plasminogen was found to greatly modify the metastatic potential in this model system; lung metastasis in plasminogen-deficient mice was significantly reduced as compared to littermate controls with respect to frequency of occurrence, total number of metastases, and total metastatic tumor burden. Plasminogen activators, as well as other key factors that govern the conversion of plasminogen to plasmin, were expressed within the mammary tumors, suggesting that the plasminogen/plasmin system may promote metastasis by contributing to tumor-associated extracellular proteolysis. The data provide direct evidence that plasmin(ogen) is a tumor progression factor in PymT-induced mammary cancer, and support the hypothesis that hemostatic factors play an important role in tumor biology.

Robust one-day in situ hybridization protocol for detection of microRNAs in paraffin samples using LNA probes

MicroRNAs (miRNAs) constitute a group of small non-coding RNA molecules generally 18-22 base-pairs in length. miRNAs are considered to be negative regulators of gene expression at the level of post-transcription and are important in normal physiological development, tissue homeostasis and disease. The cellular origin of individual microRNAs is of utmost importance for understanding their roles in molecular and biological processes, in multi-cellular and complex structured tissues. For the localization of miRNAs in clinical and experimental formalin-fixed and paraffin-embedded samples we have developed a simple and robust one-day in situ hybridization protocol based on the use of double digoxigenin (DIG)-labeled LNA-DNA chimeric probes. We show that the protocol enables analyses of specificity, and demonstrate the detection of miR-1, miR-21, miR-124, miR-126, miR-145, and miR-205 in human and murine paraffin material. The well established localization of these microRNAs makes them ideal for use as reference microRNAs when optimizing the microRNA in situ hybridization assay for a particular tissue and miRNA.

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.

The Urokinase Receptor as a Potential Target in Cancer Therapy

The glycolipid-anchored receptor for urokinase-type plasminogen activator (uPAR) is essential for cell-surface associated plasminogen activation and is overexpressed at the invasive tumor-stromal microenvironment in many human cancers. In line with this, uPAR and uPA levels in both resected tumor tissue and plasma are of independent prognostic significance for patient survival in several types of human cancer. As the expression of both uPAR and its cognate protease ligand thus appears to be correlated with tumor malignancy, the uPA-uPAR interaction represents an attractive target for the development of either antagonists with possible anti-invasive effects or cytotoxic agents with anti-tumor effects. In this review we discuss recent achievements in the development of protein and peptide based drug candidates targeting uPAR. The majority of these compounds has been optimized for human uPAR and exhibits a pronounced species-specificity showing little or no reactivity with murine uPAR. This evidently complicates their application in preclinical intervention studies, since an intimate interplay between the tumor and its associated stroma is a distinct feature of the invasive phenotype of many human cancers. The virtues and drawbacks of various mouse tumor models as surrogates for human cancer are also discussed in relation to uPAR targeting.

Stromal cells associated with early invasive foci in human mammary ductal carcinoma in situ coexpress urokinase and urokinase receptor

The transition from ductal carcinoma in situ (DCIS) of the breast to invasive ductal carcinoma is facilitated by proteolytic degradation of basement membrane. The transition can be identified as microinvasive foci in a small proportion of DCIS lesions. We have previously found that MMP‐13 is frequently expressed in such foci. To establish whether plasmin‐directed proteolysis is likely to be involved in early invasion, we have here studied the expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) in human DCIS lesions with and without microinvasion. uPA mRNA was detected in periductal stromal cells in all of 9 DCIS lesions with microinvasion and in 2 of 9 DCIS lesions without microinvasion by in situ hybridization. The uPA mRNA signal was seen in numerous stromal cells in microinvasive areas together with MMP‐13 mRNA expressing cells. Double immunofluorescence analyses, using emission fingerprinting, showed that the uPA expressing stromal cells included both myofibroblasts and macrophages. The early invasive carcinoma cells were negative for uPA. uPAR immunoreactivity was focally upregulated in periductal stromal cells in all of the 9 DCIS lesions with microinvasion and in only 2 of the 9 DCIS lesions without microinvasion. uPAR was seen in both macrophages and myofibroblasts in microinvasive areas, and it was evident that uPA and uPAR colocalized in both fibroblast‐like cells and macrophage‐like cells. We conclude that periductal macrophages and myofibroblasts are strongly involved in the initial steps of breast cancer invasion by focally upregulating the expression of the plasminogen activation system and MMP‐13.