The literature strongly implicates the fibrinolytic system, transitional fibrin deposition, and its remodeling and uPAR in particular in the pathogenesis and outcomes of a broad range of malignant neoplasms (6
). These observations suggest that remodeling of extravascular fibrin could likewise promote the growth of MPM and provide a strong rationale to more precisely define the contribution of uPAR to the pathogenesis of MPM. uPAR is a heavily glycosylated, glycosylphosphatidylinositol (GPI)-anchored membrane protein that regulates stromal remodeling by localizing uPA-mediated proteolytic activity to the cell surface. uPAR-mediated signaling can alternately influence a range of processes relevant to neoplastic growth, including cellular proliferation, migration, or invasiveness (15
). Our data extend these observations and indicate that uPAR may contribute to accelerated tumor growth and invasiveness of MPM.
To assess the ability of MPM cells differentially expressing uPAR (and other components of the fibrinolytic system) to generate intrathoracic tumors, we developed a new orthotopic model that exploits murine anatomy in that there is a shallow, medially accessible compartment anterior to the heart that is amenable to injection. Using this approach, we reliably generated intrapleural MPM tumors in nude mice with less than 5% risk of pneumothorax, which in our hands commonly occur with more lateral approaches. Although orthotopic models of intrapleural MPM have previously been reported in rats and mice, they were not used to evaluate mechanisms that underlie tumor growth (12
). Our model affords a reproducible means to interrogate pathways that promote growth of MPM and represents a potentially powerful tool for the future testing of new therapeutics. We used nude mice as hosts given our study objective, which was to compare the ability of human MPM cells with differential expression of uPAR, to generate tumors in vivo
. Because expression of other components of the fibrinolytic system and likely other effectors differed between the MPM lines, we used independent in vitro
analyses to determine the contribution of uPAR to proliferation, migratory capacity, and invasiveness.
Tumor tissues derived from the REN, MS-1, and M9K cells retained expression of appropriate markers during the progression of tumor growth and the REN cells demonstrated accelerated tumor growth in the nude athymic mice, compared with MS-1 and M9K. Extravascular fibrin was also a prominent component of the transitional stroma of the tumors derived from the REN, MS-1, and M9K cells. This situation faithfully recapitulates the findings in tumors from patients with MPM, as we previously reported (8
). Invasion of the surrounding tissues was observed in REN cell–derived tumors but not MS-1 or M9K tumors. By whole body CT scanning and postmortem analyses, the model also recapitulates the lack of distant metastases usually seen with clinical MPM.
By immunohistochemical analyses, the REN cells expressed discernibly increased uPAR versus the other cell lines. We found that uPAR expression was induced by TNF-α in these cells and that the stability of uPAR mRNA was increased in the REN cells in transcription chase experiments, extending our previously reported findings that uPAR mRNA is subject to post-transcriptional control in MS-1 and M9K cells (14
). Interestingly, uPA, PAI-1, and uPAR were all detectable by immunostaining of the excised tumors, even though the REN cells did not express uPA protein or message in culture. Consistent with these observations, increased uPAR expression was found in REN-derived tumor homogenates, while comparable levels of uPA were observed in all homogenates and PAI-1 was decreased in those derived from the MS-1 cells. We speculated that we most likely detected murine uPA within the REN stroma or associated with tumor cells tumor and confirmed that the antibody we used recognizes murine uPA. We also found that murine uPA binds REN cells but that there is little competition with soluble human uPAR for the murine enzyme. These findings raise the possibility that murine uPA binds receptors other than uPAR at the REN cell surface and that these interactions might likewise contribute to their ability to proliferate or migrate in vivo
We chose REN cells for transfection with uPAR siRNA as they expressed increased levels of uPAR and were more aggressive in vivo
. We found that proliferation and migration of uPAR-silenced REN cells was significantly impaired. Using an alternative approach, a polyclonal uPAR-neutralizing antibody that blocks the association of uPA with uPAR and likely binds other uPAR epitopes significantly decreased REN cell migration and invasion. In all, the data confirm that uPAR expression substantively contributes to the ability of REN cells to proliferate, migrate, and invade. Given that REN cells also increased proliferation and migration in response to exogenous human uPA, we conclude that the in vitro
effects are at least in part related to the association of uPA with uPAR. As we found that uPA antigen and activity was detectable in FBS and that the bovine uPA binds REN cells by flow cytometry, we further infer that bovine uPA could associate with REN cell uPAR to contribute to these effects. The differential response of the MPM cell lines in response to antibody-mediated uPAR blockade could in part be due to differences in uPA expression. uPAR can mediate both uPA-dependent and -independent migration and invasion (32
). The MS-1 and M9K cells both make uPA, whereas REN cells do not. uPAR in REN cells may mediate migration through uPA-signaling effects, whereas the localization of uPA to the cell surface may be more important for MS-1 and M9K cell migration. Thus, the uPAR antibody we used could block uPAR-mediated migration via effects on uPA–uPAR binding or uPAR-mediated signaling that vary between the different cell lines.
Given the prominent representation of extravascular fibrin in the MPM tumors in our model, we also sought to determine the ability of the MPM cells to invade three-dimensional fibrin gel matrices. Interestingly, we found that REN cells were highly migratory in this system, while the MS-1 and M9K or MeT5A pleural mesothelial cells were not. REN and MS-1 cells invaded Matrigel matrices to a comparable extent, but the increased ability of REN cells to invade fibrin may contribute to their enhanced invasiveness in vivo, given the fibrinous neomatrices we found in our MPM model. The contribution of uPAR to this response was confirmed as uPAR-silenced cell migration in fibrin matrices was significantly decreased. While we found that addition of exogenous uPA lyses the three-dimensional fibrin gel, an antibody that blocks the association of uPA with uPAR (D3036-2) interferes with migration in the fibrin gel system. These observations link uPAR expression to the differential ability of REN cells to migrate in a three-dimensional fibrin matrix, either through association with uPA present in the media or alternatively in a uPA-independent manner.
We and others have reported that uPA binding to uPAR can drive proliferation, migration, and invasion in cancer cells (34
). There are likely several mechanisms that drive these processes and that involve the localized proteolytic activity of uPA when it binds to uPAR. An alternative mechanism involves uPAR-mediated signaling after ligation with uPA that may be independent of the proteolytic activity of uPAR. The role of uPA bound to uPAR in proteolysis, invasion, and migration has been well documented in the literature (reviewed in Ref. 39
). Along those lines, the down-regulation of uPAR in human carcinoma cell lines decreases uPA-induced ERK activation and leads to tumor dormancy through disruption of a uPAR/uPA/α5β1 complex (40
). uPA has also been found to drive proliferation in cells expressing uPAR and EGFR through ERK and STAT5b activation (15
The effects of exogenous uPA were relatively muted versus that of serum, raising the possibility that uPAR mediated the effects via alternate pathways. In fact, uPAR has the ability to interact with other proteins and is known to mediate tumorogenic effects via these interactions. Resnati and colleagues reported that cleavage of uPAR by its ligand, uPA, causes a modification in uPAR which allows it to interact with the FPR-like receptor-1 receptor, FPRL1. This interaction activates the FPRL1 receptor, which then facilitates an increase in cell migration (42
). Work by Jo and colleagues further suggests that expression of uPAR by cancer cells could mediate the effects of EGF on EGFR interactions and facilitate cell proliferation. The phosphorylation of Tyr-845 and activation of STAT5b are dependent upon the presence of uPAR and EGFR and not necessarily uPA (15
). uPAR expression can also activate ERK and Rac1 in an EGFR-independent manner and thereby facilitate cell migration (44
). uPAR is also known to interact with a number of integrins, including β1 forms (reviewed in Ref. 29
), and may serve as a ligand to mediate integrin-dependent binding and signaling (45
–49). However, our data strongly suggest that the uPA–uPAR interaction could contribute, at least in part, to REN cell proliferation and migration, although other mechanisms are also likely invoked as observed when REN are stimulated with 2.5% FBS.
Due to the effects of uPAR down-regulation and blockade on classic in vitro readouts of tumor aggressiveness (e.g., decreased tumor cell proliferation, migration, and invasion), stably transfected uPAR shRNA–expressing REN cells were constructed. Only a fraction of the uPAR shRNA clones propagated to the extent that they could be assayed in subsequent analysis. The uPAR shRNA transfectant with the greatest inhibition of uPAR expression was chosen for further analysis. Unfortunately, this clone was very difficult to propagate and we were only able to obtain enough cells to inoculate three mice. A trend toward decreased volumetric tumor burden was observed in tumors generated by uPAR shRNA REN cells versus naïve or control shRNA-treated cells, but tissue invasion was observed in all tumor types. While differential patterns of invasion could emerge over more extended analyses, these experiments were not feasible. Nevertheless, the difficulty propagating uPAR shRNA–transfected REN cells themselves suggests that the receptor substantively contributes to tumor growth, and the in vivo findings are consistent with that notion. As the interpretation of these data is limited, we used an alternative approach to test the effects of stable transfection of uPAR on tumor growth of MS-1 cells.
Relatively low uPAR expressing MS-1 MPM cells were next engineered to stably overexpress uPAR. uPAR-overexpressing MS-1 cells were found to proliferate and migrate to a greater extent than naïve and EV-expressing MS-1 cells, indicating that increased uPAR expression in MS-1 cells increased MPM tumor progression in vitro. Naïve, EV-, and uPAR-overexpressing MS-1 cells were next inoculated into the pleural space of nude mice and the tumors were allowed to grow for 49 days. uPAR-overexpressing MS-1 cells were found to produce significantly more exophytic tumors and greater tumor burden than the naïve and EV-transfected cells, but evidence of tissue invasion could not be demonstrated. It is possible that the level of overexpression of uPAR was not sufficient to generate an invasive phenotype in vivo, that invasiveness might be demonstrable later in the progression of the tumor, or that uPAR is not critical to this response in MPM cells.
In summary, our data implicate uPAR in the growth of MPM cells in vitro and strongly suggest that it plays a role in accelerated tumor growth in vivo. We established a new orthotopic model of MPM which can be used to compare the growth of human MPM, evaluate underlying mechanisms responsible for tumor growth and invasiveness, or test the efficacy of new interventions. It is unclear that any interaction of murine uPA with human uPAR occurs in REN tumors in vivo or over a pathophysiologically relevant range of enzyme concentrations. uPAR-overexpressing REN cells fail to express uPA and formed tumors that grew more rapidly, were more invasive, and were more poorly tolerated than MS-1 and M9K cells that both express uPA but lesser amounts of uPAR. uPAR gene silencing and antibody blockade indicate that uPAR contributes to the ability of REN cells to proliferate, migrate, and invade. The ability of REN cells to migrate in fibrin matrices is in part related to uPAR expression, and is a potentially important property given the prominent extravascular fibrin we observed in all of the MPM tumors we studied. Further, we have established a direct link between uPAR expression and MPM tumor aggresiveness in vivo by demonstrating that increasing uPAR expression in a low uPAR expressing cell line (MS-1) increases tumor growth in nude mice. Our findings suggest that, as in other malignancies, uPAR represents a potential target for therapeutic intervention in MPM.