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Cancer cell invasion is facilitated by extracellular matrix degrading proteases such as plasmin. We have studied the expression of plasminogen activator inhibitor-1 (PAI-1) and urokinase receptor (uPAR) together with the γ2-chain of laminin-5 (lam-γ2) by immunohistochemistry in 20 cases with incipient oral squamous cell carcinoma (SCC). PAI-1-positive neoplastic cells located at the tip of the putative invasive front of grade 1 (incipient) carcinoma were seen in 16 of the 20 cases (75%), whereas adjacent normal and dysplastic epithelium was PAI-1-negative. Clusters of putative invasive neoplastic cells located in the lamina propria were PAI-1-positive in areas with grade 2 incipient carcinoma as were invasive cancer cells in areas of grade 3–4 invasive carcinoma. uPAR immunoreactivity was strongly expressed in numerous stromal cells in the carcinoma area in all 20 lesions, while a few uPAR-positive stromal cells were found in areas with normal and dysplastic epithelium. uPAR-positive neoplastic cell islands located at the front of the lesions were seen in 15 of the 20 cases. The expression pattern of lam-γ2 was very similar to that of PAI-1; however, lam-γ2-positive neoplastic cells were only detected in 11 of the 20 cases (55%) in areas of grade 1 incipient carcinoma. Direct comparison of the 3 components revealed colocalization in neoplastic cell islands in both incipient and invasive SCC. Our results suggest that PAI-1 is a novel potential marker of initial invasion in oral SCC, and that the coordinated expression of PAI-1 with uPAR and lam-γ2 sustain the features of the early invasive cancer cells. © 2006 Wiley-Liss, Inc.
Invasive squamous cell carcinomas (SCC) develop from noninvasive dysplastic precursor lesions or carcinoma in situ. The transition from noninvasive to invasive carcinoma is linked to the proteolytic destruction of the basement membrane, allowing the carcinoma cells to invade the adjacent connective tissue.1, 2, 3 Plasmin(ogen) is believed to play an important role in this process because of its capacity to degrade extracellular matrix proteins, such as fibrin and basement membrane laminin,1, 4, 5 and to activate other matrix degrading proenzymes, such as metalloproteases.6, 7 Plasminogen is an abundant enzyme in the peripheral blood and other body fluids that is converted to active plasmin by either of the 2 well-known plasminogen activators: urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). uPA-directed plasminogen activation mainly occurs on the cell surface after the binding of uPA to its specific cell-surface receptor, uPAR. This leads to focal cell-surface associated generation of plasmin, which mediates proteolysis at the cell surface required for cell migration and invasion.8 Two plasminogen activator inhibitors, PAI-1 and PAI-2, have been characterized of which PAI-1 is the primary physiologic PAI and regulates plasminogen activation in the extracellular matrix.9
The expression of uPA, PAI-1 and uPAR is upregulated in several types of cancer, including oral and pulmonary SCC and breast and colorectal adenocarcinomas.10, 11, 12, 13 The increased levels of one or more of these 3 components measured in tissue extracts or blood are associated with poor prognosis. For example, uPAR is a marker of poor prognosis in pulmonary carcinomas,14 and uPAR, uPA and PAI-1 are all indicators of poor prognosis in breast cancer.15, 16, 17 To understand the role of PAI-1 in plasminogen activation and its link to poor prognosis, nonproteolytic functions of PAI-1 should be considered. For example, the specific binding of PAI-1 to vitronectin suggests that PAI-1 also contributes to cell adhesion and migration.9, 18 Cancer cell invasion involves, in addition to the epithelial cancer cells, a variety of stromal cells (vascular cells, fibroblasts and inflammatory cells), which may contribute to the expression of the different components of the plasminogen activation system. Histological expression studies of SCC of the skin and esophagus have shown that uPA, PAI-1 and/or uPAR mRNA is predominantly expressed by the cancer cells.19, 20, 21 Immunohistochemical analyses of esophagus and head and neck SCC show a similar expression pattern,21, 22, 23 whereas the expression of the 3 components in adenocarcinomas of breast, colon and prostate is predominantly seen in the stromal compartment.24, 25, 26, 27, 28, 29, 30 Thus, the origin of the neoplasm strongly affects cell populations that contribute to the increased plasminogen activation.10, 31 Most, if not all, of the aforementioned expression studies have been performed on manifest invasive cancers, whereas little attention has been given to early carcinomas, including carcinoma in situ lesions and lesions suspected to be invasive (incipient carcinoma).
Lacking specific diagnostic markers to clearly document early invasive growth, the criteria of incipient carcinoma relies solely on the morphologic patterns of epithelial changes. To discriminate carcinoma in situ from incipient oral SCC, the integrity of the basement membrane has been considered as a valuable tool.32 However, since focal absence of basement membrane appears in (noninvasive) dysplastic lesions, laminin or collagen IV immunohistochemical staining cannot be used as reliable markers.32 For SCC of the cervix and the lower anogenital tract, the expression of the γ-2 chain of laminin-5 has been reported to be an indicator of initial invasion.33, 34 The aim of the current study is to describe the expression of PAI-1 and uPAR by immunohistochemistry in incipient oral SCC and to evaluate the significance of PAI-1 and uPAR as indicators of early invasion in oral SCC in comparison with the expression of laminin γ-2 chain.
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In this study, we have shown that the expression of 2 key regulators of plasminogen activation, PAI-1 and uPAR, is strongly upregulated in incipient and early invasive SCC of the oral cavity. The presence of normal and dysplastic epithelium as well as incipient and invasive carcinoma in most of the samples investigated allowed a semiquantitative comparison of the expression levels between these tissues, and from the analyses we concluded that the expression of PAI-1 and uPAR in epithelial cells is strongly associated with the incipient and invasive carcinoma. Our findings are based on immunohistochemistry using positive and negative control antibodies. Thus, both PAI-1 and uPAR immunoreactivity was obtained with polyclonal and monoclonal antibodies,29, 36, 37 which showed identical staining patterns and negative control antibodies that showed no specific staining. The use of paraffin embedded tissue specimens that had been fixed extensively in formalin for the current immunohistochemical studies prohibited concomitant analysis of uPA, since uPA immunoreactivity cannot be retrieved in such samples.28 However, recent immunohistochemical analyses of uPA in frozen sections from squamous cell carcinomas of the oral cavity showed the presence of uPA, primarily in cancer cells.41 The latter study also showed that the level of uPA activity is strongly increased in the malignant tissue compared to that of the adjacent normal tissue, a finding that substantiated earlier observations of increased uPA mRNA levels in oral SCC.42, 43 Taken together, these results indicate that uPA-directed plasminogen activation is a strongly active process during cancer progression in the oral cavity, the strength of which may be directly related to early recurrence for some of these patients.13
We have subdivided the epithelial changes into areas with apparently nonmalignant mucosa (normal and hyperplastic/dysplastic epithelium), areas suspected to be invasive (incipient carcinoma) and areas with unambiguous invasion, according to Anneroth et al.35 Using this classification, we found that PAI-1 was upregulated in areas corresponding to grade 1 incipient carcinoma in 16 of the 20 cases (75%) investigated as well as in all higher grades, whereas PAI-1 was virtually absent in normal, hyperplastic and dysplatic epithelium. Similarly, lam-γ2 was detected in 11 of the 20 cases (55%) in areas with grade 1 carcinoma and all higher grades. The expression pattern of uPAR was found to be more complex by being expressed in various cell types and not confined to the carcinoma areas. These findings suggest that PAI-1 expression in incipient oral carcinoma is the most effective indicator of initial invasion in oral SCC compared to uPAR and lam-γ2. A larger study of patients with suspected invasive oral SCC analyzed in a follow-up study would be required to determine whether the immunohistochemical detection of PAI-1 can be used as a clinical parameter to discriminate noninvasive from early invasive oral SCC.
We found strong PAI-1 immunoreactivity in the neoplastic cells of incipient carcinoma and invasive carcinoma, but virtually no PAI-1 in the adjacent stromal cells, which is in contrast to the expression pattern found in breast, colon and prostate cancer where PAI-1 is expressed mainly in myofibroblasts.26, 29, 30 Immunohistochemical staining of PAI-1 in oral squamous cell carcinomas has also been reported by Nozaki et al.23 and Yasuda et al,22 who also observed PAI-1 immunoreactivity in cancer cells, but only in ˜30 and 80% of their cases, respectively. Yasuda et al.22 analyzed cryoembedded samples of mainly grade 3 and 4 oral SCC for PAI-1 and, in agreement with our findings, found PAI-1 immunoreactivity at the surface of cancer cell islands (the basal cancer cells). However, these authors also found PAI-1 immunoreactivity in the extracellular matrix in close proximity to the cancer cell islands that interestingly colocalized with vitronectin immunoreactivity, a major binding partner for PAI-1. In our study, PAI-1 immunoreactivity was only observed in cytoplasm of neoplastic cells and a few stromal fibroblast-like cells, while we found only discrete extracellular PAI-1 positive debris in parts of the stroma close to the incipient and invasive carcinoma. This subtle disagreement is likely to be explained by the fact that Yasuda et al.22 analyzed frozen sections. Alternatively, this difference may be explained by the specificity of the pAb used. The pAb employed by Yasuda et al.22 were raised against PAI-1 purified from bovine endothelial cells and affinity purified on a similar PAI-1 preparation, allowing the possibility of co-purifying crossreacting antibodies. In contrast, we used pAb obtained by immunization with PAI-1 purified from HT1080 cells and subsequently affinity purified against recombinant human PAI-1 expressed yeast.29
The expression pattern of PAI-1 was found to be considerably different from that of uPAR. The highest level of uPAR immunoreactivity was observed in all cases in stromal cells associated with incipient and invasive SCC. In addition to the intensely stained stromal cells, we observed clusters of uPAR-positive neoplastic cells in 15 of the 20 cases generally found as invasive cancer cells located in the most profound areas of the invasive lesions. Thus, uPAR was detected in subpopulations of cancer cells and was not expressed by all cancer cells in any of the cases. These findings disagree with those reported by Nozaki et al.,23 who observed uPAR immunoreactivity only in cancer cells. Notably these authors employed the avidin-biotin detection system and detected uPAR only in approximately one third of their cases, whereas we used the more sensitive envision detection system44 and detected uPAR in all cases.
Our observations indicate a complex expression pattern for uPAR in both incipient and definite invasive oral SCC as it is expressed in various cell populations, including macrophages, neutrophils, (myo)fibroblasts and cancer cells, the predominant expression being observed in cells located in the stromal compartment. Predominant expression of uPAR immunoreactivity in stromal cells has been described in various adenocarcinomas, including colon, gastric and hepatocellular carcinoma,24, 45, 46 whereas uPAR immunoreactivity in SCC of the esophagus21 and uPAR mRNA in SCC of the skin20 was predominantly observed in the cancer cells.
Despite the dissimilarity in the expression patterns of uPAR and PAI-1, we observed a noteworthy correlation of their expression in neoplastic cells in areas of incipient as well as invasive SCC. Interestingly, this coexpression also coincided with the expression of the γ2 chain of laminin-5. Laminin-5 is an extracellular protein that links the basement membrane via integrins to hemidesmosomes. However, this interaction is sensitive to proteolytic cleavage mediated by MT1-MMP, which releases domain III of the lam-γ2 chain and subsequently leads to cell motility.47 Interestingly, the keratinocytes upregulate lam-γ2, uPAR and PAI-1 during healing of skin wounds,48, 49, 50 indicating that these molecules are involved in the migratory and invasive phenotype of the leading-edge keratinocytes. Therefore, the concomitant expression of PAI-1 and frequently also of uPAR and lam-γ2 in the same focal microenvironment and even in the same oral neoplastic cells strongly suggests a sustained invasive capacity of these cells. Coexpression of uPAR and lam-γ2 has been reported in invasive cancer cells in human colon adenocarcinoma,39 but apparently these cancer cells do not express PAI-1.26 This divergence may either be explained by the different types of epithelia in the two locations (simple epithelium in the intestine vs. stratified squamous epithelium in the oral cavity) or the unique character of the local mesenchymal environment.
The high expression of lam-γ2 in neoplastic cells of incipient and invasive carcinoma along with its general absence in normal oral mucosa, suggests that lam-γ2, like PAI-1, is a potential marker of initial invasion in oral squamous cell carcinoma. Indeed, lam-γ2 is an established marker of invasion in squamous cell carcinomas of the cervix and the vulva.33, 34 The transition from noninvasive to invasive carcinoma is likely to be mediated by the activity of one or several extracellular matrix-degrading proteases expressed in the local microenvironment,2 such as it has been described in carcinoma in situ lesions of the human breast, where MMP-13 is expressed in myofibroblasts in connection with early invasion.51 In conclusion, our results together with those of Curino et al.41 indicate that the early steps in invasive growth of oral squamous cell carcinomas are associated with the expression of the plasminogen activation cascade. Our findings suggest that PAI-1 is a marker of malignancy in incipient carcinomas of the oral cavity, and that the coordinated expression of PAI-1, uPAR and lam-γ2 could be a molecular profile acquired for early invasion in oral SCC.