Combined mRNA expression levels of members of the urokinase plasminogen activator (uPA) system correlate with disease-associated survival of soft-tissue sarcoma patients

Soft-tissue sarcomas (STS) are malignant mesenchymal neoplasias with an incidence of about 1% among all human malignancies [1]. STS enlarge leading to the appearance of a pseudo capsule composed of an inner compression zone and an outer reactive zone at formation of fingers, which give rise to satellite lesions several centimeters away from the primary tumor [2]. The major clinical problems in the treatment of STS are the propensity of the tumor to recur locally, and the fact that many patients without obvious clinical metastases harbor occult micro-metastases that become clinically evident. Lymph node metastases are rare in STS patients [3, 4]. Despite adequate local control of the primary tumor, about 50% of sarcoma patients will succumb to distant metastatic disease [5].

The urokinase plasminogen activator (uPA) system has been shown to play a major role in the pericellular network of interacting proteolytic systems that are able to degrade extracellular matrix components and facilitate tumor invasion and metastasis [6, 7]. Furthermore, components of the uPA system have been implicated in proliferation, migration and adhesion of tumor cells as well as in tumor angiogenesis [8, 9]. Components of the uPA system, which consists of the serine protease uPA, its receptor uPAR and its principal inhibitor PAI-1 are prognostic factors in different types of cancer. High antigen levels of uPA and/or PAI-1 protein in tumor tissue extracts are strong predictors of poor prognosis in patients afflicted with different types of solid malignant tumors including sarcomas [10, 11]. High uPAR levels are also associated with poor prognosis in various cancer types, however, the prognostic impact of uPAR expression is not as pronounced as that of uPA and PAI-1 [12, 13]. In contrast, the expression of a mRNA splice variant of wild-type uPAR (uPAR-wt) lacking exons 4 and 5 (uPAR-del4/5) has been demonstrated to be a highly sensitive, independent prognostic marker in breast cancer patients [14–16].

Whereas wild-type uPAR consists of three structurally homologous domains, in the uPAR-del4/5 variant the complete domain II of uPAR is deleted, and the uPAR-del4/5 protein does not interact with either of its ligands uPA or vitronectin [17]. However, in breast cancer cells the overexpression of the uPAR-del4/5 protein profoundly affects the in vitro invasion capacity of cells through a Matrigel matrix, the adhesion to extracellular matrix proteins and also lung colonization in an in vivo metastasis model. These observations strongly suggest that uPAR-del4/5 displays biological activity modulating tumor biological relevant processes [17].

In sarcomas, high expression of uPA and uPAR antigen as detected by immunohistochemistry has been reported to be an independent prognostic factor for metastasis-free survival and overall survival in chondrosarcoma patients [18]. In soft-tissue sarcomas, increased uPA protein levels in tumor tissue were found to significantly correlate with local recurrence and metastasis in a cohort of 69 STS patients [19]. We recently reported a highly significant correlation between high antigen levels of uPA, PAI-1 or uPAR in tumor tissue, and of soluble uPAR in serum, with poor outcome of STS patients [20]. However, only one report has investigated the mRNA expression of components of the uPA system in tumor tissue from soft-tissue sarcoma patients [21]. In this study, which included 38 STS patients, significantly higher uPA, PAI-1 and uPAR mRNA expression levels were observed in tumor tissue compared to paired adjacent normal tissue. Additionally, PAI-1 mRNA expression was found to be 6-fold higher in metastatic tumors than in non-metastatic tumors [21].

In the present study, we determined the mRNA expression levels of uPA, PAI-1 and uPAR in a cohort of 78 adult STS patients and analyzed their relationship with prognostically relevant clinical and histomorphological parameters and disease-associated survival. Furthermore, the impact of mRNA expression of uPA system components on disease-associated survival was evaluated in clinically relevant subgroups of STS patients with complete (R0) or non-radical (R1) tumor resection.

The uPA system is one of the best-investigated protease systems in both physiological and pathological conditions, including cancer [reviewed in [6, 7]]. In clinical studies, high protein contents of uPA and/or PAI-1 in tumor tissue have been shown to indicate an unfavorable prognosis in various types of cancer [10, 11, 26]. For example, the determination of antigen levels of uPA and PAI-1 in tissue extracts of primary breast cancer has entered clinical practice for risk-adapted, individual therapy decisions in patients with lymph node-negative disease [11, 26]. In soft-tissue sarcoma, however, few studies have analyzed the protein expression of uPA system components and evaluated the impact of expression of these proteins on prognosis of STS patients. Choong et al. [19] reported that increasing uPA protein levels in tumor tissue were associated with local recurrence and metastasis in 69 STS patients. Recently, high protein levels of uPA, PAI-1 and uPAR in tumor tissue extracts were found to be significantly associated with a shortened disease-associated survival of STS patients; this association was independent of prognostically relevant clinical parameters [20]. Moreover, combined values of high uPA, PAI-1 or uPAR tissue levels with high serum levels of soluble uPAR were highly significantly and independently associated with poor disease-associated survival; these patients showed an approximately 6-fold increased risk of tumor-related death [20].

Generally, measurements of tumor biological markers in tissue extracts by ELISA require relatively large amounts of fresh-frozen tumor material as well as adequate storage capacities for tumor samples. Furthermore, tumor tissue material is more limited when the cancer is diagnosed at early stages because most tumor tissue specimens are retrieved from fine needle aspirates or core biopsies [27]. Thus, alternative, less material-consuming methods for the quantitative determination of prognostic factors in tumor tissue such as quantitative PCR are desirable.

In the present study, we analyzed the mRNA expression levels of uPA system components in a cohort of 78 patients with soft-tissue sarcoma, and high correlations were found between the mRNA values of uPA, PAI-1, uPAR-wt and an uPAR splice variant, uPAR-del4/5 (with r_s values greater than 0.70). In agreement with these results, positive correlations between mRNA values of uPA system components have been observed in several other studies [23, 28–31]. At the protein level, similar significant correlations between uPA, PAI-1 and uPAR antigen levels in tumor tissue have been reported [20, 23, 30, 32, 33]. These findings may not be surprising because various interactions of the members of the plasminogen activation system may affect each other or are regulated in a concerted manner in tumor growth and metastasis.

In contrast, we found only low/moderate correlations between uPA, PAI-1 and uPAR-wt mRNA expression and the respective antigen levels in tumor tissue from the STS patient cohort (r_s values ranging from 0.26 to 0.35; data not shown). Other studies have also indicated that uPA and/or PAI-1 antigen levels in tumor tissue do not completely reflect the respective mRNA expression level [23, 28, 30, 34, 35]. This discrepancy could be due to posttranscriptional regulation processes, which have previously been reported for components of the uPA system [36].

Although mRNA levels do not completely mirror antigen contents in tumor tissue, a number of studies have demonstrated that mRNA expression levels of certain uPA system members are linked with clinical and histomorphological parameters, and/or prognosis in cancer patients. In breast cancer, Witzel et al. [35] have reported that patients with high PAI-1 mRNA expression have a reduced 10-year disease-free survival and overall survival rate, and PAI-1 mRNA expression might reveal additional clinically relevant information compared to PAI-1 protein levels. In other studies that have analyzed PAI-1 mRNA expression in different sets of breast cancer patients, high PAI-1 mRNA levels were found to be significantly associated with shorter metastasis-free or overall survival, whereas uPA mRNA levels had no prognostic relevance [37, 38]. However, in a subset of ErbB2-positive breast cancer patients, Urban et al. [29] have identified uPA mRNA as the most significant marker associated with distant metastasis-free survival, whereas PAI-1 mRNA was found to be significantly associated with distant metastasis-free survival, independent of the ErbB2 status. In addition, high uPA and PAI-1 mRNA levels were found to be significantly associated with shorter disease-free survival in primary breast cancer patients, independent of hormone receptor or lymph node status [28]. In other tumor types, such as pancreatic and gastric cancer, mRNA expression of uPA system components has also been found to be related to prognosis [31, 39–41]. However, the existence of studies in which no relationship was found between uPA, PAI-1 or uPAR mRNA expression and patients' survival is notable [30, 42].

Other studies have also examined uPA system components in soft-tissue sarcomas. In a small cohort of 38 STS patients, significantly higher PAI-1 mRNA expression levels, but not uPA or uPAR levels, were observed in metastatic compared to non-metastatic tumors [21]. In the present study, uPA, PAI-1 and uPAR-wt mRNA values did not differ significantly between tumors in relation to clinical and histomorphological parameters, except for a significant relationship between high PAI-1 mRNA expression and tumor grade and between PAI-1 and uPAR-wt mRNA levels and the histological STS subtype, which has also been reported by others [21]. Interestingly, mRNA levels of uPAR-del4/5, an uPAR mRNA splice variant described recently [14], were significantly correlated with clinical prognostic factors such as histological subtype, tumor grade and tumor stage in STS patients. However, the expression levels of uPAR-del4/5 mRNA, which has recently been shown to be strongly associated with prognosis in lymph node-negative breast cancer patients [16], had no prognostic impact in STS patients. Similarly, mRNA expression of the other uPA system markers did not show a significant correlation with disease-associated survival in the whole cohort of STS patients.

However, when we analyzed the expression pattern of uPA system markers in the subgroup of STS patients who underwent radical tumor resection (R0) we observed a trend towards an association between mRNA expression and disease-associated survival. Specifically, elevated mRNA values of uPAR-del4/5 and PAI-1 were associated with a 6-fold increased risk of tumor-related death in R0 patients (but not in R1 patients). It is tempting to speculate that increasing the number of patients in this subgroup analysis would result in significant associations between high levels of uPAR-del4/5 or PAI-1 mRNA levels and disease-associated survival. Moreover, by using combined PAI-1 and uPAR-del4/5 mRNA expression values, R0-resected patients with high PAI-1/uPAR-del4/5 mRNA values had a significant, 19-fold increased risk of tumor-related death compared to R0-resected patients who displayed low PAI-1/uPAR-del4/5 expression levels. These results suggests that the quantitative determination of PAI-1 and uPAR-del4/5 mRNA expression levels in R0-resected STS patients provides additional prognostic information that may allow for individual, risk-adapted (adjuvant) therapy decisions. Thus, R0 patients at high risk, as identified by elevated uPAR-del4/5 and/or PAI-1 mRNA levels, may benefit from chemotherapy, which would result in the reduction of tumor progression. In contrast, patients displaying low mRNA expression of both uPAR-del4/5 and PAI-1 in tumor tissue could be spared the exposure to chemotherapy.

In the present study, we analyzed mRNA expression levels of uPA, PAI-1, uPAR and an uPAR mRNA splice variant, uPAR-del4/5, in a cohort of 78 adult soft-tissue sarcoma patients. Our data suggest that mRNA levels of PAI-1 and uPAR-del4/5 are significantly related to clinical and histomorphological parameters of STS patients such as histological subtype, tumor grade or tumor stage. In addition, in STS patients with complete tumor resection high PAI-1 or uPAR-del4/5 mRNA levels are associated with a distinctly shortened disease-associated survival of STS patients. Strikingly, co-detection of a high mRNA level of uPAR-del4/5 and PAI-1 in tumor tissue of STS patients with complete tumor resection is associated with a 19-fold increased risk of tumor-related death suggesting that both markers can be considered together for prognostic evaluation and may support future therapy stratification.