1 Department of Surgery, 2 Department of Pathology, 3 National Research Laboratory for Cancer Epigenetics, 4 Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea; 5 Laboratory of Cell Regulation and Carcinogenesis, National Cancer Institute, Bethesda, MD, USA
Received 30 August 2003; revised 26 November 2003; accepted 22 December 2003
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ABSTRACT |
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Transforming growth factor-ß (TGF-ß) modulates the growth and function of many cells, including those with malignant transformation. Smad proteins have been identified as major components in the intracellular signaling of TGF-ß family members.
Patients and methods:
To clarify the correlations between clinicopathologic profiles and the patients survival, the expression of common mediator Smad (Smad4) and inhibitory Smad (Smad7) were evaluated immunohistochemically in 304 consecutive gastric carcinomas using the tissue array method.
Results:
Positive Smad4 expression was observed in 266 (87.5%) tumors and positive Smad7 expression in 98 (32.2%) tumors. The prognosis of patients with a Smad4-positive tumor was significantly better than that of the patients with a negative tumor. The survival rate was significantly higher in patients with negative Smad7 expression than those with positive Smad7 expression. In subgroup analysis according to TNM (tumournodemetastasis) stage, both Smad4 and Smad7 showed most significant prognostic differences in stage I gastric cancer patients. Multivariate analysis indicated that tumor size, depth of invasion, lymph node metastasis and Smad7 expression were independent prognostic factors.
Conclusion:
Enhanced expression of the TGF-ß signaling inhibitor Smad7 may present one of the novel mechanisms of TGF-ß resistance in human gastric carcinomas.
Key words: gastric carcinoma, prognostic factor, Smad4, Smad7, transforming growth factor-ß
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Introduction |
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The gene encoding Smad4 was originally cloned as a tumor suppressor gene on chromosome 18q21, which is frequently deleted or mutated in pancreatic carcinomas. Hence, its original name was DPC4 (deleted in pancreatic carcinoma locus 4) [7]. Smad4 is also mutated in a significant proportion of colorectal tumors and less frequently in breast, ovarian, head and neck, prostatic and esophageal cancers [812]. Recent studies of human clinical samples have suggested that decreased expression of Smad4 or LOH (loss of heterozygosity) at 18q21 may be associated with a more aggressive phenotype in gastric, esophageal and pancreatic cancers, and intrahepatic cholangiocarcinoma [1316]. Xiangming et al. have previously reported that reduced expression of Smad4 was related to the depth of tumor invasion and reduced Smad4 expression was an unfavorable prognostic factor for advanced gastric cancer [17].
The inhibitory Smads, Smad6 and Smad7, have been shown to bind to the TGF-ß type I receptor, precluding the phosphorylation of the receptor-regulated Smads and, consequently, interfering with TGF-ß signaling [18]. It has been reported previously that Smad7 is overexpressed in pancreatic cancer, suggesting that this is a mechanism of repression of the TGF-ß-signaling pathway [19]. More recently, the position of the gene for Smad7 has been assigned to the region 18q21, identical to Smad4, by in situ hybridization and mapped between Smad2 and Smad4 genes with a 4-Mb gene cluster [20, 21]. However, little is known about the roles of Smad7 in gastric carcinoma.
The purpose of this study was to investigate the expression of Smad4 and Smad7 proteins and their prognostic significance in human gastric carcinoma.
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Materials and methods |
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Immunohistochemistry
Immunohistochemical staining against Smad4 [1:100; B-8, sc-7966 (Santa Cruz Biotechnology, Santa Cruz, CA)] and Smad7 [1:100; H-79, sc-11392 (Santa Cruz Biotechnology)] was performed using a streptavidin peroxidase procedure. After deparaffinization and rehydration, tissue sections were treated three times with microwaves in 0.01 M citrate buffer (pH 6.0) for 5 min each time. The sections were then immersed in methanol containing 0.3% hydrogen peroxidase for 6 min to block the endogenous peroxidase activity, and incubated in 2.5% blocking serum to reduce non-specific binding. After incubation with primary antibodies, the sections were incubated with biotinylated anti-rabbit IgG and avitinbiotin peroxidase (Vector Laboratories, Burlingame, CA), and visualized using diaminobenzidine tetrahydrochloride. Two antibodies among various commercially available antibodies were selected after the test procedure using a human control slide for immunohistochemistry (Superbiochips Laboratories).
For statistical analysis, the results of immunostaining were considered to be positive if 10% of the neoplastic cells were stained. Previous studies in which immunolabeling patterns have been correlated with Smad4 gene status have shown that both focal and diffusely positive labelings correlate with an intact Smad gene, whereas complete loss of labeling correlates with inactivation of the Smad gene [22]. On the contrary, there have been few studies dealing with the definite level of Smad7 positivity in immunohistochemical staining, so we applied the previously reported methodology on Smad7 positivity in immunohistochemical staining [23]. For purposes of data analysis, both focal and diffusely positive lesions were considered to show intact Smad expression (positive), and only complete loss of labeling was considered to show loss of Smad expression (negative).
Statistical analysis
The association of factors was evaluated using the chi-square test. The significance of differences among means was determined by the MannWhitney U-test. Survival rates were calculated using the KaplanMeier method and analyzed using the Log-rank test. A multivariate analysis was performed using the Cox proportional hazards model. The significance level was set at 5% for all analyses. All statistical analyses were conducted using the SPSS 10.0 statistical software program (SPSS, Chicago, IL).
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Results |
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Correlation between Smad4 or Smad7 expression and survival rate
The 5-year survival rate was 64.4% in patients with Smad4-positive tumors and 49.8% in patients with Smad4-negative tumors. Accordingly, the prognosis for patients with a Smad4-positive tumor was significantly better than for patients with a negative tumor (P = 0.017). In subgroup analysis according to TNM (tumournodemetastasis) stage [24], the survival rate of patients with a Smad4-positive tumor was significantly higher than that of patients with a Smad4-negative tumor with stage I (93.3% versus 77.9%; P = 0.037) and IV (21.6% versus 0%; P = 0.05), but there were no statistical significances in the tumors with stage II (65.3% versus 87.5%) and III (40.7% versus 45.5%) (Figure 2). Moreover, the 5-year survival rate of patients with tumors in which Smad7 expression was positive was 52.2%, whereas the survival rate of patients with tumors in which Smad7 expression was negative was 67.5%. The survival rate was significantly higher in patients with negative Smad7 expression than in patients with positive Smad7 expression (P = 0.011). In subgroup analysis according to TNM stage, the survival rate of patients with a Smad7-negative tumor was significantly higher than that of patients with a Smad7-positive tumor with stage I (95.6% versus 84.1%, P = 0.025) and III (50.0% versus 26.1%, P = 0.05), but there were no statistical significances in the tumors with stage II (66.6% versus 72.2%) and IV (17.2% versus 16.7%) (Figure 3).
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Discussion |
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In this study, we examined Smad4 and Smad7 expression in gastric carcinomas to elucidate their role in tumor progression. The tissue array method was used to analyze Smad4 and Smad7 proteins in 304 consecutive gastric carcinomas. The tissue array method enabled us to analyze a large number of gastric carcinomas, and consecutive sections from the array blocks allowed different protein expressions to be analyzed from defined, almost morphologically identical, tumor regions.
The Smad4 gene, located at 18q21, has been found to undergo frequent alteration in pancreatic cancers [7] and LOH studies have suggested that a gene on chromosome 18q is altered frequently in intestinal-type gastric carcinomas [27]. In our study, the loss of Smad4 protein expression was also statistically significantly associated with intestinal type, and associated with male sex, larger tumor size, differentiated tumors, deep penetration, advanced clinical stage and patient survival. Xiangming et al. reported that the reduced expression of Smad4 was related to the depth of tumor invasion and that Smad4 was an independent prognostic factor. Therefore, mutation in the Smad4 gene and loss of Smad4 protein either resulted in tumorigenesis or was associated with malignancy and progression of tumors [17]. Our results also revealed that the loss of Smad4 expression was related to the deep penetration and poor survival, but Smad4 was not an independent prognostic factor in multivariate analysis in our series [28]. These different results may be attributable to the selection of patients and different cut-off positive value, or to the evaluation of the results.
Smad7 inhibits TGF-ß-induced transcriptional responses [29]. Smad7 associates with the activated TGF-ß receptor and interferes with the activation of Smad2 and Smad3 by preventing their receptor interaction and phosphorylation. It has been reported that Smad7 acts as an important molecule for regulating TGF-ß activity in human disease. Monteleone et al. reported that Smad7 was overexpressed in irritable bowel disease mucosa and purified mucosal T cells [30]. In a separate study, Kleeff et al. reported that Smad7 enhances tumorigenicity in pancreatic cancer [19]. Using in vitro and in vivo studies, they revealed that pancreatic cancer cells have redundant barriers to TGF-ß signaling that may allow the cancer cells to escape TGF-induced growth inhibition, while still allowing for the expression of metastasis-promoting genes such as PAI-1. But, until recently, whether Smad7 expression is associated with clinicopathological parameters such as tumor stage and prognosis has not been reported. In this study, Smad7 expression was significantly more frequent in intestinal type and differentiated carcinoma. The underlying etiology of this tumor heterogeneity is not well understood. The survival time analysis revealed a significant correlation between Smad7 expression and length of disease-free survival. The prognostic value of Smad7 was independent of other well established clinical prognostic factors such as depth of invasion or nodal involvement. We conclude that Smad7 plays an important role in the development of gastric carcinoma and that overexpression of Smad7 may be a significant independent prognostic indicator for clinical outcome in patients with gastric carcinoma.
In subgroup analysis according to TNM stage, although Smad4 (stage IV) and Smad7 (stage III) showed some marginal prognostic significance, both Smad4 and Smad7 showed the most significant prognostic differences only in stage I gastric cancer patients. The reason why Smad4 and Smad7 expression should be most prognostic only in stage I patients is unclear, but in case of Smad4-negative stage I gastric cancer patients, all patients were classified as having stage Ib disease (eight T2N0M0 and one T1N1M0). In the clinical setting, this result could be applied very usefully for selecting the patients who should be followed closely and considered as candidates for adjuvant treatment among patients with stage I gastric cancer.
As mentioned previously, Smad4 and Smad7 are both located on chromosome 18q21. Therefore, it would be of considerable interest to know whether gene expression of Smad4 and Smad7 were independent of each other. However, our results showed that the expression patterns of Smad4 and Smad7 were inversely correlated with each other (P = 0.04), which suggests that these two closely located genes might be expressed by a different mechanism.
As expected, comparing co-expression of Smad4 and Smad7 with survival, the 5-year survival rate for patients with Smad4+/Smad7 expression was most favorable, while that with Smad4/Smad7+ expression was most unfavorable [significant difference (P = 0.001)]. The 5-year survival rates with Smad4/Smad7 and Smad4+/Smad7+ expression were found to lie in between. Therefore, evaluating the expression profile of both Smad4 and Smad7 together could be a more useful prognostic marker for gastric cancer patients than evaluating only one expression profile of either Smad4 or Smad7.
In summary, this study has demonstrated that Smad7 expression is associated with poor outcome in gastric carcinomas. These observations further underscore the importance of Smad proteins in carcinogenesis, and indicate that Smad7 expression may present one of the novel mechanisms for TGF-ß resistance in human gastric carcinoma. As both Smad4 and Smad7 showed most significant prognostic differences in stage I gastric cancer patients, Smad expression in gastric cancer could be useful in selecting the patients who should be closely followed and considered as candidates for adjuvant treatment among those with stage I gastric cancer.
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Acknowledgements |
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FOOTNOTES |
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