Ann Thorac Surg 2005;79:248-253
© 2005 The Society of Thoracic Surgeons
Original article: General thoracic
Prognostic Significance of the Tumor Suppressor Gene Maspin in Non–Small Cell Lung Cancer
Kyoji Hirai, MDa,*,
Kiyoshi Koizumi, MDa,
Shuji Haraguchi, MD,
Tomomi Hirata, MDa,
Iwao Mikami, MDa,
Mitsuhiro Fukushima, MDa,
Shigeki Yamagishi, MDa,
Tetsuo Kawashima, MDa,
Daisuke Okada, MDa,
Kazuo Shimizu, MDa,
Masashi Kawamoto, MDb
a Department of Surgery II
b Department of Pathology, Nippon Medical School, Tokyo, Japan
Accepted for publication June 25, 2004.
* Address reprint requests to Dr Hirai, Department of Surgery II, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan (E-mail: ky-hirai{at}nms.ac.jp).
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Abstract
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BACKGROUND: Maspin is a serpin protease inhibitor, which is known to suppress tumor progression in breast cancer and to be regulated by wild-type p53. This study was performed to elucidate the biologic significance of maspin expression in non–small cell lung cancer.
METHODS: To investigate whether maspin is involved in progression, clinicopathologic features, and prognosis of non–small cell lung cancer, we performed an immunohistochemical study using antimaspin antibody and identified the presence of maspin messenger ribonucleic acid in cancerous and noncancerous tissues by reverse transcription–polymerase chain reaction analysis. In addition, we evaluated p53 expression immunohistochemically on the serial sections.
RESULTS: Most adenocarcinoma and squamous cell carcinoma showed cytoplasmic staining pattern. The cytoplasmic positive rate was 77.8% (42 of 54 specimens) for the stage III group, and 36.2% (21 of 58 specimens) for the stage I group (p < 0.0001). Three-year survival rates after operation were 30.8% for the maspin-positive group and 71.1% for the maspin-negative group (p = 0.007). In multivariate analysis, immunohistochemical maspin expression in patients with non–small cell lung cancer was an independent prognostic factor for overall survival. No correlation between maspin and p53 expression in cancer cells could be observed. There was an average fourfold increase in maspin messenger ribonucleic acid levels in cancerous tissues compared with those of noncancerous tissues, and stage III cases exhibited significantly higher maspin messenger ribonucleic acid levels than stage I cases (p = 0.003).
CONCLUSIONS: The results of this study suggest that overexpression of maspin in cytoplasm may be a useful marker of tumor progression and unfavorable prognosis for overall survival in some patients with non–small cell lung cancer. Furthermore, maspin expression in cytoplasm appears to be unaffected by p53.
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Introduction
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Maspin (mammary serpin) is a serine protease inhibitor, the member of the serpin family with an amino acid identity closest to that of equine and human neutrophil-monocyte elastase inhibitors (43% and 39%, respectively), human plasminogen activator inhibitor type 2, human squamous cell carcinoma antigen, and chicken ovalbumin [1]. Its gene, which is a part of the serpin locus cluster at chromosome 18q21.3q23, encodes a 42-kDa cytoplasmic protein with sequence homology to other inhibitory serpins [2, 3]. Maspin protein is located at the cell membrane and the extracellular matrix [4]. It has been shown to regulate plasminogen activation by a single-chain tissue plasminogen activator [5]. As for tissue plasminogen activator, aside from a few reports suggesting that it is involved in cell growth [6], cell motility, and invasion [7], its function in epithelial cell biology and tumorigenesis remains unknown.
The maspin gene was identified in normal mammary epithelium. In another report, the presence of maspin in several normal epithelia (prostate, thymus, testis, small intestine, colon) has been shown [8]. In contrast, tumor cells exhibited a decreased expression or absence of maspin. In particular, maspin in breast cancer suppressed tumor progression in vitro and tumor cell metastasis in vivo [9]. On the other hand, maspin is an angiogenesis inhibitor, which prevents endothelial cells from forming tubes and completely blocks basic fibroblast growth factor–induced neovascularization [10]. The downregulation of maspin with increasing malignant potential is controlled at the transcriptional level [11]. A recent report has demonstrated that its mechanism is closely associated with cytosine methylation and chromatin condensation [12]. Furthermore, it has been shown that upregulation of maspin leads to the tumor inhibitory effects of manganese-containing superoxide dismutase [13].
In the present study, we investigated the localization of maspin protein and messenger ribonucleic acid (mRNA) in non–small cell lung cancer (NSCLC) to determine whether maspin expression plays an important role in tumor progression and clinical outcome. We also sought to clarify the correlation between maspin and p53.
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Material and Methods
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Tumor Specimens and Survival Data
Tumor specimens from 132 patients were obtained by surgery at Nippon Medical School between 1999 and 2001. The patients consisted of 90 men and 42 women (average age, 65.5 ± 9.2 years at diagnosis). Histopathologically, tumor specimens were diagnosed as adenocarcinoma (n = 94) and squamous cell carcinoma (n = 38). They represented 58 pathologic stage (p-stage) I, 20 p-stage II, and 54 p-stage III. The p-stage was determined according to the guidelines of the American Joint Committee on Cancer. Staging was determined on the basis of the tumor, node, and metastasis (TMN) classification of the International Union Against Cancer. The clinicopathologic features of the patients and tumors are summarized in Table 1.
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Table 1. Relationship Between Maspin Expression and Clinicopathologic Features and p53 Expression in 132 Non–Small Cell Lung Cancer Patients
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Immunohistochemistry
All of the samples were collected in compliance with requirements of the Institutional Review Board for the Protection of Human Subjects. Formalin-fixed, paraffin-embedded tissues from lung cancers and normal lung tissues were cut into 3-µm-thick sections. After microwave-based antigen retrieval with 0.05 mol/L Tris buffer (pH 9.0) for 15 minutes, the sections were incubated with the anti–human maspin monoclonal antibody (PharMingen International, San Diego, CA; diluted 1:75) for 1 hour and the anti–human p53 monoclonal antibody (DO 7; DAKO, Tokyo, Japan; diluted 1:100) for 30 minutes. The LSAB kit (DAKO) was used as the detection system. Color was produced by using 3,3'-diaminobenzidine plus substrate (DAKO) for 1 minute. Slides were counterstained with Mayer's hematoxylin for 5 minutes. We counted 500 of cancer cells. All immunostained sections were examined by at least two pathologists. Positive staining was defined as the presence of cytoplasmic maspin and p53 immunoreactivity in at least 40% of cancer cells.
Reverse Transcription–Polymerase Chain Reaction
Total ribonucleic acid (RNA) was prepared from unfixed frozen specimens of primary NSCLC and normal tissues. Extraction of total RNA from the homogenized specimens was carried out with Isogen (Nippon Gene, Tokyo, Japan). Complementary deoxyribonucleic acid (cDNA) was synthesized using RNA polymerase chain reaction (PCR) kit (AMV Ver. 2.1, TAKARA, Tokyo, Japan). Using this cDNA as the template DNA, reverse transcription (RT)-PCR was performed for carcinomas and normal lung tissues remote from carcinomas, which was resected from 40 cases (15 stage I cases, 10 stage II cases, 15 stage III cases). The human maspin PCR primers were 5'-GAA CGA CCA GAC CAA AAT CC-3' (sense) and 5'-CAA TCT TCT CCA AGC CTG TG-3' (antisense). Human ß-actin PCR primers were used as an endogenous control and were 5'-AAA TGC TTC TAG GCG GAC TA-3' (sense) and 5'-CTG GGC CAT TCT CCT TAG AG-3' (antisense). The PCR was performed using a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). Electrophoresis of the PCR products on a 2% agarose gel revealed maspin bands of approximately 281 base pairs (bp) and ß-actin bands of approximately 343 bp. Each band was analyzed by National Institutes of Health image analysis software version 1.56 (Bethesda, MD). The density values of maspin amplification products were normalized to those of ß-actin. For each type of PCR analysis, the PCR products from a different number of cycles (15 to 40) were analyzed. The results from the 30-cycle program used here were on the linear portion of the amplification curves.
Statistics
Statistical analysis of the experimental results in immunohistochemical analysis was obtained by Fisher's exact or 
2 tests. Survival curves were generated using the Kaplan-Meier method, and differences in survival were analyzed by the Wilcoxon test. Multivariate analysis for prognostic value in overall survival was analyzed with the Cox proportional hazards regression model. The results were considered significant if the p value was less than 0.05. All calculations were carried out using the Statview computer program (Ver. 5.0, SAS Institute Inc, Cary, NC).
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Results
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The expression and localization of maspin were examined in lung cancers using anti–human monoclonal antibody. In normal bronchial gland epithelial cells around lung cancer cells, maspin showed weak immunoreactivity around the nucleus; however, no expression of maspin could be found in normal alveolar epithelial cells as well as in stromal cells (Fig 1A). Most of the cancer cells in adenocarcinoma and squamous cell carcinoma showed a strong cytoplasmic staining pattern for maspin (Figs 1B, 1C). Maspin immunoreactivity was absent in the stromal cells around cancer cells. The relationship between maspin and p53 expression of lung cancers was evaluated in serial sections.
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Fig 1. Immunohistochemical staining patterns for maspin and p53 in non–small cell lung cancer and normal lung tissue. (A) Weak immunostaining for maspin surrounding the nucleus of bronchial gland epithelial cells in normal lung tissue and negative staining in normal alveolar epithelial cells (magnification, x200). (B, C) Strong immunostaining for maspin in cytoplasm of adenocarcinoma and squamous cell carcinoma, and negative staining of stromal cells (magnification, x200).
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Maspin immunostaining of 132 lung cancers and their associated clinicopathologic features are summarized in Table 1. Maspin-positive rate of cancer cells was determined to be 55.3%. There was no significant difference in the percentages of maspin positivity between the two histologic types (Table 1). As for differentiation, maspin positivity of cancer cells was found in 57.4% of well-differentiated types, 59.5% of moderately differentiated types, and 44.4% of poorly differentiated types. There was no significant association regarding differentiation. No significant difference could be found in tumor classification. Maspin expression was associated with lymph node involvement (p = 0.011, N0 versus N2 and N3). In addition, stage I cases exhibited significantly higher positivity for maspin than stage III cases (p < 0.0001). The assessment of p53 and cytoplasmic maspin expression in serial sections of lung cancers is also summarized in Table 1. Maspin expression in cytoplasm had no correlation with p53 expression (p = 0.786).
Reverse transcription–PCR analysis using maspin-specific oligonucleotide primers and ß-actin primers as an intrinsic control showed the presence of maspin and ß-actin mRNA in cancerous and distal noncancerous tissues. The representative findings in the three cases are shown in Figure 2. The RT-PCR products from maspin mRNA were semiquantified using ß-actin as an intrinsic control. The density of each band was separately converted to a numeric value by National Institutes of Health image analysis software. The ratio of maspin to ß-actin in each case was calculated. The average maspin/ß-actin value in cancerous tissues was approximately fourfold that in normal tissues (1.15 ± 0.63 versus 4.64 ± 2.66; p < 0.0001; Fig 3A). Furthermore, the average ratio of tumorous to nontumorous tissue (T/N ratio) of the maspin/ß-actin was 3.89 in stage I cases and 6.68 in stage III cases, respectively (p = 0.003; Fig 3B).
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Fig 2. Reverse transcription–polymerase chain reaction analysis of maspin and ß-actin messenger ribonucleic acid in normal lung tissues and cancer tissues of 3 different patients.
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Fig 3. (A) Maspin/ß-actin ratio from semiquantification of reverse transcription–polymerase chain reaction products in normal tissues and cancer tissues (mean ± standard deviation). (B) Comparative tumor/nontumor (T/N) ratios for maspin messenger ribonucleic acid (mean ± standard deviation) in stage I (n = 15), stage II (n = 10), and stage III (n = 15).
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To evaluate whether cytoplasmic maspin expression has prognostic significance in lung cancer, we analyzed the survival of patients using the Kaplan-Meier method. The overall survival curve for the patients is shown in Figure 4. Three-year survival rates after operation were 30.8% for the maspin-positive group and 71.1% for the maspin-negative group. Statistical analysis showed that patients with maspin overexpression had a survival rate that was significantly shorter than those who were maspin-negative (Wilcoxon test, p = 0.007; Fig 4). Table 2 summarizes the multivariate analysis of prognostic value in overall survival in 132 NSCLC patients. In this study, a significant value in overall survival was observed for lymph node involvement (p = 0.004) and maspin expression (p = 0.034).
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Fig 4. Kaplan-Meier survival of non–small cell lung cancer patients on the basis of maspin immunoreactivity using the Wilcoxon statistic.
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Comment
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Maspin was introduced as a tumor suppressor gene of breast and prostate malignancies associated with cell motility and invasion [14]. Originally, maspin is expressed in normal human mammary epithelial cells [1]. Its loss has been linked with metastatic potential and tumor progression, which is regulated by the prominent suppressor gene, wild-type p53 [15]. A single-chain tissue plasminogen activator interacts with the maspin-reactive site loop peptide and forms a stable complex with the recombinant maspin, suggesting that maspin may have an inhibitory effect on tissue plasminogen activator [5]. However, the mechanism underlying the function of maspin is not completely understood. According to the immunohistochemical study of maspin in oral squamous cell carcinoma, high maspin expression was associated with the absence of lymph node involvement and epithelial differentiation [16]. Collectively, it was considered that increased maspin expression was not beneficial to tumor function. An updated report has demonstrated that cytoplasmic maspin is overexpressed in a substantial proportion of ovarian tumors, and may be used as an unfavorable prognostic factor [17]. In another recent report, a high frequency of maspin in pancreatic tumors was detected; however, in all normal pancreatic tissues its lack of expression was found [18]. In addition, Syed and associates [19] demonstrated that increased maspin in cytoplasm was significantly associated with a poor prognosis. As suggested in these three reports, a new biologic significance for cytoplasmic maspin is imminent.
Previously, the presence of maspin in both the nucleus and cytoplasm has been pointed out [8]. Although maspin nuclear staining in breast cancer was significantly correlated with a favorable prognosis, maspin was predominantly present in the cytoplasm of tumor cells [17]. In view of these findings, some possible explanations and implications were examined; however, definite evidence for a paradoxic mechanism remains unelucidated. To determine the relationship of the two components (nuclear and cytoplasmic maspin), maspin expression in other organs needs to be evaluated in detail.
In this study, we investigated the association between cytoplasmic maspin and p53 in serial sections of NSCLC using immunohistochemical analysis to compare clinicopathologic data. We provide evidence in NSCLC that the higher rate of maspin expression in stage III (77.8%) compared with that in stage I cases (36.2%) is significant, and that the increased cytoplasmic maspin expression is linked to poor prognosis. Furthermore, analysis of maspin expression and clinicopathologic features demonstrated that high maspin expression was associated with the existence of lymph node involvement (p = 0.002). Our study showed that maspin was not associated with age, sex, histologic type, or histologic differentiation. In all normal bronchial gland epithelial cells, a weak localization of maspin around the nucleus was observed. In cancerous tissues, tumor cells showed a strong diffuse cytoplasmic pattern and a partially weak nuclear staining pattern; however, no positive findings could be observed in stromal cells. Cytoplasmic maspin may be inactive, and it is possible that the nuclear localization of maspin, which plays a critical role in biologic function, represents the active form. In addition, according to our results that immunoreactivity in normal bronchial epithelial gland cells was significantly weaker than that in tumor cells, cytoplasmic maspin may also be a useful marker of the carcinogenesis of NSCLC. We also elucidated that an increase in maspin mRNA expression was frequent in human cancerous tissues from patients with NSCLC. These findings were consistent with the results of immunohistochemical analysis (data not shown).
The tumor-suppressor gene p53 is located on chromosome 17q13.1. The function of this gene is, as a nuclear protein, the control of cell cycle activation, apoptosis, and maintenance of genomic stability. p53 appears to be the most frequent genetic defect identified in human cancers, including lung cancer [20]. Although wild-type p53 is involved in the secretion of thrombospondin 1, an angiogenesis inhibitor, mutant p53 downregulates thrombospondin mRNA and increases vascular endothelial growth factor. As described previously, maspin is an angiogenesis inhibitor, which blocks endothelial migration induced by vascular endothelial growth factor. In addition, Zou and colleagues [15] have demonstrated that p53 may suppress tumor metastatic potential by enhancing KAI1 (suppression of tumorigenicity 6, prostate; CD82 antigen [R2 leukocyte antigen, antigen detected by monoclonal and antigen IA4]), PAI1 (plasminogen activator inhibitor 1), and maspin, providing that wild-type p53 directly regulates maspin expression. Disadvantageous effects on tumor function may be dependent on the inhibitory mechanism of angiogenesis associated with both p53 and maspin. Machtens and coworkers [21] demonstrated that in prostate cancer decreased maspin in the nucleus is correlated with positive immunoreactivity for p53. In contrast, in our immunohistochemical study, we showed no relationship between maspin and p53 expression. This result suggests that in some organs cytoplasmic maspin may be expressed without correlation to abnormal accumulation of p53, which may indicate a paradoxic role of cytoplasmic maspin in ovarian carcinoma and pancreatic cancer [17, 18]. Further investigation of cytoplasmic maspin in various cancers is required.
In conclusion, our results suggest that maspin overexpression in the cytoplasm of NSCLC is significantly associated with tumor progression. This is the first study to show that the expression of maspin in cytoplasm is an independent prognostic factor for NSCLC patients. Additionally, increased maspin in cytoplasm may predict a poor prognosis, and may be expressed without correlation to abnormal accumulation of p53.
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Abstract
Introduction
