The Tumor Cell Surface-Associated Urokinase-Type Plasminogen Activator
January 1, 2001 7 minutes read
The Tumor Cell Surface-Associated Urokinase-Type Plasminogen Activator as a Maspin Target
By Shijie Sheng, PhD
In a search for tumor suppressor genes, the mammary serine protease inhibitor (maspin) gene was identified by subtractive hybridization on the basis of its expression at the mRNA level in normal, but not tumor-derived, breast epithelial cells.1
Maspin protein has an overall sequence homology with members of the serine protease inhibitor (serpin) superfamily.1 Subsequent studies show an inverse correlation between maspin expression and breast cancer progression and oral squamous cell carcinoma, as well as head and neck cancer.1-5 In addition, maspin expression is down-regulated in prostate carcinoma cells compared with that in immortalized normal prostate epithelial cells.6 Thus, the down-regulation of maspin expression appears to be an important attribute to tumor progression toward more aggressive phenotypes. Biological evidence further suggests the potential therapeutic application of maspin in treating human malignancies. Maspin specifically inhibits the invasion and motility of an array of breast and prostate carcinoma cells in vitro, and inhibits the growth and metastasis of human breast and prostate cancer cells in xenograft mouse models.1,6-9
The Mechanism of Maspin
To develop maspin-based, therapeutic anticancer strategies, it is crucial to understand the molecular mechanism of maspin. Previous in vitro studies using both endogenously expressed maspin or purified recombinant maspin protein have demonstrated that maspin’s inhibitory effect on tumor invasion and metastasis is, at least in part, due to its localized action at the interphase between cell membrane and the extracellular matrix (ECM).6,10,11 As reported earlier by Seftor and associates, one of the downstream events following maspin treatment of breast carcinoma cells MDA-MB-435 was an increased cell surface expression of a3b1 integrin, which in turn led to increased cell adhesion to fibronectin.11 However, to date, the direct molecular target of maspin remains elusive. Central to this issue, maspin does not act as a classical serpin in cell-free solutions; i.e., it does not inhibit a series of serine proteases including tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA).12
In 1998, Sheng and colleagues reported that maspin specifically inhibits tPA associated with fibrinogen or polylysine.12 Based on the complex interaction between maspin and the fibrinogen-associated tPA, it was hypothesized that maspin may specifically target plasminogen activators that are bound to a biological surface such as the plasma membrane. It is well documented that uPA, but not tPA, binds specifically to its cell surface-anchored receptor, uPAR.13,14 Furthermore, uPA along with uPAR is causatively involved in the invasive and metastatic phenotypes, and the poor prognoses of many types of carcinomas.15,16 Thus, it is important to determine whether a potential inhibitory interaction between maspin and the tumor cell-associated uPA underlies the molecular mechanism of maspin.
Maspin and Tumor Cell Surface-Associated uPA
A recent report by McGowen coworkers provided the first evidence that the tumor cell surface-associated uPA is a maspin target.17 To address the specific interaction between maspin and the cell surface-associated uPA, the authors took advantage of the established prostatic carcinoma cell line DU145 that produces uPA as the predominant plasminogen activator, but does not express detectable levels of endogenous maspin. In addition, the authors developed a coupled enzymatic assay to detect the plasminogen activation mediated by viable DU145 cells in monolayer culture. It was shown that recombinant human maspin protein specifically inhibited DU145 cell-mediated uPA activity. The inhibitory effect of maspin was similar to that of a specific uPA-neutralizing antibody, and was reversed by the polyclonal antibody made against the reactive site loop (RSL) sequence of maspin. Based on a current paradigm, an inhibitory serpin uses its RSL to dock into the catalytic site of the target enzyme. This initial docking induces a massive b-sheet rearrangement of the serpin molecule, leading to a stabilized enzyme/inhibitor complex.18,19 The essential role of the intact RSL of maspin in its proteolytic inhibitory activity toward cell-associated uPA suggests that maspin may act as an inhibitory serpin.
Interestingly, as compared to several serpin inhibitors of plasminogen activators, such as PAI-1 and PAI-2, maspin exhibited a novel cell surface-dependent interaction with uPA. First, under a non-permeabilizing condition, maspin bound to the surface of DU145 cells in a saturable manner, suggesting that it may interact specifically with a cell surface-associated molecule. Second, maspin inhibited only the cell surface-associated uPA, not the secreted uPA or purified uPA in cell-free biochemical reactions. Third, maspin formed a stable complex only with the uPA in the cell lysate fraction of DU145 cells, not with the purified uPA or the uPA secreted by DU145 cells into the conditioned culture medium. These data demonstrated an important role for the epithelial cell surface in mediating the inhibitory interaction between maspin and uPA. The authors postulated that since the activation and the activities of uPA on the cell surface are mediated by its receptor uPAR, the association with uPAR may render uPA prone to inhibition by maspin.13,14 On the other hand, the cell surface microenvironment also may provide additional cofactors that further increase the inhibitory potency of maspin by facilitating its critical transition from a latent to an active conformation.
McGowen et al also performed detailed kinetic analyses, showing that maspin acted as a competitive inhibitor of uPA with an apparent Ki value of 20 nM. These kinetic characteristics are comparable to those of PAI-1 and PAI-2 in similar cell-based biochemical analyses. It is important to point out, however, that different plasminogen-activator inhibitors may play distinct roles in tumor progression. For example, PAI-1, along with uPA and uPAR, is causatively involved in the progression of breast cancer.20 In contrast, maspin, which is down-regulated in several types of carcinomas, has tumor-suppressing activity. In their report, McGowen et al further demonstrated that the proteolytic inhibitory effect of maspin was quantitatively consistent with its inhibitory effect on the motility of DU145 cells in vitro.
Conclusion
Taken together, the results of McGowen et al suggest that maspin may inhibit tumor cell invasion and motility by blocking the uPA-mediated, ECM-degrading proteolytic cascade. While future studies are needed to test whether endogenous maspin protein has a similar proteolytic inhibitory effect on the cell surface-associated uPA, and whether uPA produced by other types of carcinoma cells is inhibited by maspin, it is intriguing to hypothesize that novel maspin-based, therapeutic strategies may prove useful to target specific human malignancies that are associated with markedly elevated uPA. (Dr. Sheng is an Assistant Professor of Pathology in the Department of Pathology, Wayne State University School of Medicine, Detroit, MI.)
References
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