© The Author 2006. Published by Oxford University Press.
EDITORIAL |
Conflicting Results from Clinical Observations and Murine Models: What Is the Role of Plasminogen Activators in Tumor Growth?
Affiliation of authors: Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, Bethesda, MD
Correspondence to: Giovanna Tosato, MD, Building 10, 12C205, 10 Center Dr., Basic Research Laboratory, Center for Cancer Research, National Cancer Institute Bethesda, MD 20892 (e-mail: tosatog{at}mail.nih.gov).
Tissue invasion and metastasis can compromise vital body functions and are the main causes of death from cancer. By invading locally and dislodging cells or fragments to distant sites, primary tumors can access a rich supply of oxygen and nutrients that is limited at the primary site. Invasion and metastasis are exceedingly complex and incompletely understood multistep processes. Often, they are accompanied by the activation of proteases, which degrade proteins that tether tumor cells to surrounding cells and extracellular matrix. By changing the physical coupling of tumor cells to their microenvironment, proteases are thought to promote local cancer spread and formation of metastases (1–3). Proteases can also promote tumor angiogenesis (4) and stimulate tumor cell growth (5,6).
Invasive tumor cells and reactive stromal cells recruited to the tumor site secrete proteinases such as matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs), urokinase-type plasminogen activator (uPA) and its inhibitors (PAIs), and cathepsins. Many studies have focused on the precise identification of these enzymes, evaluation of their contribution to tumor pathogenesis, predictive prognostic value, and their potential as therapeutic targets. Considerable progress has been made. We now know, for example, that many MMPs and TIMPs secreted by cancer cells can facilitate tumor invasion and that elevated serum TIMP-1 levels are associated with poor cancer survival (7,8). We also know that active enzymes of the cysteine cathepsin family localize to the invasive front of tumors and to the angiogenic tumor vasculature (9). Importantly, we have learned from several large studies that high tumor levels of uPA and its inhibitor PAI-1 are poor prognostic factors for patients with breast cancer (10–12) and gastrointestinal, lung, brain, kidney, bladder, and soft tissue tumors (11,13–15).
uPA and the related enzyme tissue-type plasminogen activator (tPA) are serine proteinases that catalyze the cleavage of plasminogen into plasmin. uPA is primarily involved in tissue degradation and tPA in thrombolysis. Plasmin enzymatically digests the extracellular matrix (ECM) components laminin and fibronectin and can activate other proteases such as collagenase (16). uPA is secreted as an inactive single-chain proenzyme. It binds to cell surface glycolipid-anchored receptor and is then converted to an active two-chain molecule by several enzymes, including plasmin. uPA activity is regulated by two uPA inhibitors, PAI-1 and PAI-2, which bind to active uPA and promote its internalization and degradation.
In this issue of the Journal, Merchan et al. (17) examined the effects of forced expression of uPA and tPA on the growth of a murine mammary carcinoma cell line. The authors report that tumors derived from injection of cells overexpressing uPA or tPA grew slower into the mammary fat pad and produced fewer lung metastases than tumors generated by control cells and that the mice bearing uPA- or tPA-overexpressing tumors survived longer than the controls. In vitro, the carcinoma cells transduced with uPA or tPA grew at a similar rate as control cells, indicating that uPA and tPA did not directly regulate cell proliferation. Unlike tumor cells that produce active uPA, tumor cells that overexpress enzymatically inactive forms of uPA were similar to control cells in their tumorigenicity, suggesting that protease activity is required to reduce tumor growth. Thus, the results of Merchan et al. appear at odds with clinical observations that link high tumor expression of uPA with poor prognosis, and they prompt several considerations.
What are the differences between the experimental mouse tumor model presented by Merchan et al. and human cancer? To more closely mimic human breast cancer, the authors overexpressed the murine uPA gene in a murine mammary cancer cell line (BALB/c strain) and inoculated the cells orthotopically into immunocompetent BALB/c mice. Nonetheless, critical differences may exist between the model and the human disease. First, are enzyme levels achieved in the experimental system comparable to those in the human cancer? The authors have generated clones that overexpress the enzyme, in spite of the fact that the mammary carcinoma 4T1 cells express endogenous plasminogen activator (PA) mRNAs. Enzyme levels critically influence biological activities. Overproduction of uPA in endothelial cells was incompatible with normal capillary morphogenesis, which requires an appropriate balance between proteases and their inhibitors (18). At superphysiologic concentrations, PAI-1 inhibited tumor invasion and neovascularization, but tumor growth was reduced in PAI-1 null mice (19,20). Second, the timing of enzyme expression in relation to tumor development may differ. The human correlative studies used tissue specimens from primary surgery (12,13), and we do not know how enzyme levels fluctuated from tumor outset. In the experimental model of Merchan et al., high-level protease activity was present from the time the tumor cells were inoculated, which may have impaired initial cell attachment and growth (21,22). Indeed, Merchan et al. (17) showed that, in vivo, PA-overexpressing tumor cells proliferated less than control cells from a very early time point. Third, the site of enzyme release may differ. Tissue specimens of human breast cancer from patients and from a xenograft model showed that expression of uPA is strongest in the stromal fibroblasts at the invasive front of the tumors (20,23), whereas in the model described by Merchan et al., high PA expression in tumors was diffuse. Thus, enzyme levels, time of release, and location of release may constitute critical differences between the experimental tumor model of Merchan et al. and the human disease. Finally, we do not know the mechanisms underlying the antitumor activity of PAs in the experimental model, particularly the potential role of the antiangiogenic protein fragments angiostatin, endostatin, and tumstatin, which are generated by uPA.
Results from phase III clinical trials of broad-spectrum MMP inhibitors have been disappointing and instructive (24). The strong association between high-level expression of MMPs and tumor progression provided confidence that MMP inhibitors might be effective anticancer drugs. Yet, none of the trials with MMP inhibitors showed increased patient survival (24). We now know that besides their ability to promote tumor growth through ECM degradation, MMPs exert antitumor activities by degrading and inactivating chemokines that mediate tumor cell metastasis and by inhibiting tumor angiogenesis (25). Importantly, not all MMPs seem good targets for inhibition in the cancer setting. For example, mice null for MMP-3, -8, or -9 display enhanced tumorigenicity in some models (25–27), and clinical trials with tanomastat (BAY12–9566, Bayer), which blocks MMP-2, MMP-3, and MMP-9 to a greater extent than other MMPs, showed a worse clinical outcome than standard treatment, and the trial was stopped (28). Thus, some MMPs may be considered anticancer agents, and broad MMP inhibition as a form of anticancer treatment is being reevaluated (6,25). Could uPA inhibitors be developed as anticancer agents? The clinical results linking high-level uPA to poor cancer prognosis are solid and important; but what is the reason for the apparent paradox that uPA and its inhibitor PAI-1 are both independently associated with poor cancer prognosis? The study by Merchan et al. (17) supports the contention that high levels of uPA at the tumor site could be beneficial. However, the study relies on a single cell line and few clones and raises important unresolved questions relative to the role of PAs in cancer progression. Future work aimed at addressing these questions will likely yield additional insight into the invasive behavior of cancer and may predict whether PAs or their downstream mediators could become validated targets or candidate drugs for cancer.
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