Maspin expression in normal lung and non-small-cell lung cancers: cellular property-associated expression under the control of promoter DNA methylation

Abstract Maspin has been demonstrated to be a suppressor of invasion and cell motility in vitro, whereas in vivo analyses have reported that increased expression of maspin is associated with malignant behavior. The present study examined maspin expression in normal lung and non-small-cell lung cancers. Only proximal airway cells in the normal lung expressed maspin, and the expression was associated with decreased methylation. This association was also observed in non-small-cell lung cancers, but the expression was quite different among histologic subtypes; 20 of 21 squamous cell carcinomas showed intense, uniform expression, whereas the expression status varied among adenocarcinomas. Of the 119 adenocarcinomas, 60 were negative, 23 positive and 36 showed a heterogeneous expression pattern. The expression was inversely correlated with markers of peripheral airway cells. Taken together, the results suggest that maspin may be expressed in association with the proximal airway cell type. It is of note that the heterogeneous expression pattern of maspin is quite distinctive, showing geographic positivity in the individual tumors. Separate analysis of methylation status in positive and negative portions of individual tumors provided an instance of intratumor diversity associated with promoter DNA methylation. Introduction The mammary serine protease inhibitor maspin was first isolated by Zou et al. (1994) as a defective molecule in breast carcinoma cells by differential display analysis. Maspin exhibits significant homology to the serpin superfamily of serine protease inhibitors, which includes the plasminogen activator inhibitors 1 and 2 (PAI-1 and PAI-2), and α1-antitrypsin. Recent analyses in vitro have suggested an inhibitory effect on tumor invasion and metastasis. Cell motility and invasion are inhibited with transfection of the maspin gene into cancer cell lines, and the transplantation of the transfectants in nude mice led to reduced tumorgenicity and decreased metastatic potential (Sheng et al., 1994; Zou et al., 1994; Sheng et al., 1996; Zou et al., 2000). The mechanism underlying maspin's inhibitory activity remains controversial, but recent reports suggested that it does not directly inhibit matrix-degrading proteases, but rather functions as a regulator of plasminogen-tissue-type plasminogen activator complex (Bass et al., 2002). On the other hand, it has been shown that maspin has two consensus p53-binding sequences in its promoter region, and p53 regulated maspin expression, indicating that maspin is one of the target genes of p53 pathway (Zou et al., 2000). Furthermore, maspin was shown to have an inhibitory effect on tumor angiogenesis (Zhang et al., 2000) and a sensitizing effect on apoptosis (Jiang et al., 2002). Despite a tumor suppressing role in vitro, clinicopathological analysis using in vivo tumors failed to demonstrate the role, and maspin-expressing tumors tend to show more malignant behavior, including shorter survivals, in breast cancers (Umekita et al., 2002; Bieche et al., 2003), pancreatic cancers (Maass et al., 2001) and ovarian cancers (Sood et al., 2002). Promoter DNA hypermethylation is one of the epigenetic mechanisms to silence certain genes. So far, a number of genes have been shown to be inactivated by this gene silencing (Jones and Baylin, 2002; Laird, 2003), whereas the silencing was predominantly reported in the cancer tissues, but not in normal tissues. Although the involvement of DNA methylation in X-chromosome inactivation (Mohandas et al., 1981) and genomic imprinting (Li et al., 1993) has been widely accepted, tissue-specific regulation of gene expression in normal tissues mediated by DNA methylation has long been speculated. Recently, Futscher et al. (2002) first demonstrated that the tissue-specific expression of maspin was controlled by DNA methylation. They described a close correlation between maspin expression and the absence of DNA methylation using various normal tissues, and this expression in immortalized cells was restored by treatment with 5-aza-2′-deoxycytidine. In addition to normal tissues, the gene silencing of maspin was observed in breast cancers (Domann et al., 2000; Maass et al., 2002), suggesting a contribution to breast carcinogenesis. Lung cancers, especially adenocarcinomas, are characterized by a high degree of morphological heterogeneity, which in turn implies both intra- and intertumor diversities. We have been interested in and have analysed the diversities. Recently, we revealed that thyroid transcription factor-1, TTF-1, serves as a lineage marker for peripheral airway cells, including type I and II pneumocytes (Yatabe et al., 2002). Furthermore, analysis of various cancer-associated genes, including p53 (Nishio et al., 1997), cyclin D1 (Nishio et al., 1997), RB (Nishio et al., 1997), p27Kip1 (Yatabe et al., 1998b) and COX1 (Yatabe et al., 1998a; Achiwa et al., 1999), suggests a different molecular pathway for carcinogenesis in lung adenocarcinomas between cells with and without TTF-1 expression. This result implies that one of the intertumor heterogeneities of lung adenocarcinoma is represented by putative original cells, that is, peripheral airway cell-derived carcinomas and the others. This distinction is supported by the expression profiling analysis (Bhattacharjee et al., 2001; Garber et al., 2001). Unsupervised hierarchical clustering, based on the molecular signature, classified lung adenocarcinomas largely into two subtypes, which were delineated by TTF-1. The present study also focused on the diversities. First, we confirmed cell-type-specific expression of maspin, and then we examined lung tumors, revealing that maspin is expressed in a highly heterogeneous fashion in lung adenocarcinomas, similar to the morphology. We found that the expression of maspin is associated with cell type in lung tissue, while intratumor diversity of maspin is associated with regional promoter hypermethylation. There are few references in the literature concerning DNA methylation associated with the intratumor diversity (Graff et al., 2000; Nass et al., 2000; Markl et al., 2001), and this study therefore provides new information regarding maspin expression, which might shed light on the complex mechanism of the metastatic process. Results Cell-type-specific expression of maspin in the normal lung Tissue-specific expression, including airway epithelium, has been previously reported (Futscher et al., 2002), but there are various types of the epithelium covering airway tracts. Therefore, we further examined which cell types in the airway epithelium expressed maspin. Immunohistochemical analysis demonstrated a characteristic expression pattern, and this expression was restricted to the basal cells of the bronchial epithelium (Figure 1 (a1)) and myoepithelium of the bronchial glandular acini (Figure 1 (a2)). This was in sharp contrast to the peripheral portion of the lung parenchyma; none of the cells (...truncated)