Jiang PH, Motoo Y, Garcia S, Iovanna JL, Pébusque MJ, Sawabu N. Down-expression of tumor protein p53-induced nuclear protein 1 in human gastric cancer. World J Gastroenterol 2006; 12(5): 691-696 [PMID: 16521180 DOI: 10.3748/wjg.v12.i5.691]
Corresponding Author of This Article
Yoshiharu Motoo, MD, Department of Medical Oncology, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan. motoo@kanazawa-med.ac.jp
Article-Type of This Article
Gastric Cancer
Open-Access Policy of This Article
This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Pei-Hong Jiang, Norio Sawabu, Department of Internal Medicine and Medical Oncology, Cancer Research Institute, Kanazawa University, Kanazawa, Japan
Yoshiharu Motoo, Department of Medical Oncology, Kanazawa Medical University, Ishikawa, Japan
Stéphane Garcia, Juan Lucio Iovanna, Marie-Josèphe Pébusque, INSERM U624, Marseille, France
ORCID number: $[AuthorORCIDs]
Correspondence to: Yoshiharu Motoo, MD, Department of Medical Oncology, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan. motoo@kanazawa-med.ac.jp
Telephone: +81-76-218-8284 Fax: +81-76-218-8283
Received: May 30, 2005 Revised: May 30, 2005 Accepted: June 1, 2005 Published online: February 7, 2006
Abstract
AIM: Overexpression of tumor protein p53-induced nuclear protein 1 (TP53INP1) induces G1 cell cycle arrest and increases p53-mediated apoptosis. To clarify the clinical importance of TP53INP1, we analyzed TP53INP1 and p53 expression in gastric cancer.
METHODS: TP53INP1 and p53 expression were examined using immunohistochemistry in 142 cases of gastric cancer. The apoptosis of gastric cancer cells was analyzed using the TUNEL method. The relationship between the expression of TP53INP1 and clinicopathological factors was statistically analyzed.
RESULTS: TP53INP1 was expressed in 98% (139/142 cases) of non-cancerous gastric tissues and was down-expressed in 64% (91/142 cases) of gastric cancer lesions from the same patients. TP53INP1 expression was significantly decreased (43.9%) in poorly differentiated adenocarcinoma compared with well or moderately differentiated adenocarcinoma (81.6%). Cancers invading the submucosa or deeper showed lower positively (59.1%) compared with mucosal cancers (85.2%). Decrease or loss of TP53INP1 expression was significantly correlated with lymphatic invasion (54.3% vs 82.0% without lymphatic invasion) and node-positive patients (31.3% vs 68.3% in node-negative patients). P53 was expressed in 68 (47.9%) patients of gastric cancer, whereas it was absent in normal gastric tissues. A significant association was also observed between TP53INP1 status and the level of apoptosis in tumor cells: the apoptotic index in TP53INP1-positive tissues was significantly higher than that in TP53INP1-negative portions. Finally, when survival data were analyzed, loss of TP53INP1 expression had a significant effect in predicting a poor prognosis (P = 0.0006).
CONCLUSION: TP53INP1-positive rate decreases with the progression of gastric cancer. TP53INP1 protein negativity is significantly associated with aggressive pathological phenotypes of gastric cancer. TP53INP1 is related to the apoptosis of gastric cancer cells. The decreased expression of the TP53INP1 protein may reflect the malignant grade of gastric cancer and is regarded as an adverse prognostic factor.
Citation: Jiang PH, Motoo Y, Garcia S, Iovanna JL, Pébusque MJ, Sawabu N. Down-expression of tumor protein p53-induced nuclear protein 1 in human gastric cancer. World J Gastroenterol 2006; 12(5): 691-696
Tumor protein 53-induced nuclear protein 1 (TP53INP1) is a p53-inducible gene encoding two protein isoforms able to modulate p53 biological activities[1-4]. TP53INP1 expression is strongly induced in vivo in mice with acute pancreatitis[1], and in vitro in several cell lines submitted to various stress agents[2,4]. Over-expression of TP53INP1 induces cell cycle arrest in G1 phase and enhances the p53-mediated apoptosis[3]. TP53INP1 co-localizes with p53 and the serine-threonine p53-kinase HIPK2[5] within the promyelocytic leukemia protein nuclear bodies (PML-NBs) and physically interacts with these proteins modifying the p53 transcriptional activity on several p53 target genes[3]. TP53INP1 thus appears as a key-element in p53-mediated cell death and cell cycle arrest, induced by cellular stresses. The multi-step model of carcinogenesis in gastric cancer, the second most common cancer leading to death in the world, suggests accumulation of genetic alterations, epigenetic changes and posttranslational modifications. It often metastasizes to other organs, including the liver, lung, and ovary[6]. Multiple factors are known to be related to gastric carcinogenesis, including Epstein-Barr virus[7] and H pylori infections[8], microsatellite instability[9]. From the molecular point of view, it has now been established that gastric carcinogenesis is involved the accumulation of mutations in oncogenes and tumor suppressor genes controlling epithelial cell growth and differentiation[10-14]. In particular, TP53 mutations are frequently seen in gastric cancers and correlates with gastric cancer prognosis[15,16]. However, the molecular alterations and their role in gastric cancer still remain to be fully defined.
Previous works implied that TP53INP1 is a pro-apoptotic gene induced by p53[2]. Overexpression of TP53INP1 promotes G1 arrest and apoptosis through the p53-mediated pathway[3]. The aim of the present study was to analyze the expression patterns of TP53INP1 in a large series of gastric carcinomas to (1) identify the possible modulation of TP53INP1 expression; (2) investigate the association with apoptotic activity; (3) analyze the relationship with clinicopathologic parameters, and evaluate its prognostic value.
MATERIALS AND METHODS
Patients and specimens
One hundred and forty-two patients with gastric cancer were enrolled in this study. The areas adjacent to cancer lesions were used as non-malignant gastric tissue. The patients underwent operation at the Cancer Research Institute Hospital, Kanazawa University. The histological classification was defined using the Japanese classification of gastric carcinoma[17]. Intestinal type was defined as either papillary or well to moderately differentiated tubular adenocarcinoma. Diffuse type was defined as poorly differentiated adenocarcinoma, signet-ring cell carcinoma, or mucinous adenocarcinoma. The series included 104 men and 38 women, and the mean age was 63.1±10.6 years. There were 76 and 66 cases of differentiated and undifferentiated type, respectively.
Immunohistochemistry
A standard avidin-biotin-peroxidase complex method (ABC) was used for immunostaining. Deparaffinized sections were treated by microwaving at a high power for 5 min twice in a 10 mmol/L citrate buffer to retrieve antigenicity. After washing with PBS, the sections were immersed in 3% hydrogen peroxide in methanol for 20 min to block any endogenous peroxides activity. Then the ABC staining system kit (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was used for the detection. Sections were incubated with 10% normal serum for 1 h to inhibit nonspecific antibody binding. Then, sections were incubated overnight at 4 °C with 6 µg/mL of rat anti-human monoclonal antibody raised against TP53INP1 (kindly provided by Carrier). After washing with PBS, detection was done by successively incubating the sections with biotinylated goat anti-rat IgG for 30 min, and avidin-biotin-HRP for 30 min. After extensive washings with PBS, sections were stained with 3,3-diaminobenzidine for 2-10 min. Then, sections were counterstained with hematoxylin, dehydrated, and mounted. Nuclei were lightly counterstained with Mayer’s hematoxylin. TP53INP1-positive cells were counted in fields chosen at random (100× magnification), and the percentage of the number of positive cells per 1 000 cells was expressed as TP53INP1-positive index (%). Using the same method we counted the TP53INP1-positive in nucleus and in cytoplasm under the microscopy with a 200× magnification. The normal IgG was used as a negative control.
Immunohistochemistry for p53 was performed using a DAKO LSAB kit (DakoCytomation, Kyoto, Japan). The primary antibody was mouse monoclonal antibody against human p53 (DO-7, Nichirei Inc., Tokyo, Japan). The procedure was according to the protocol from the company. Finally the sections were incubated with DAB substrate as a chromogen. The cell nuclei were also lightly counterstained with Mayer’s hematoxylin.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)
TUNEL-positive epithelial cells were detected in the sections using ApopTag Plus peroxides in situ apoptosis detection kit (Chemicon International, Inc., Temecula, CA, USA). Briefly, after pretreatment with 20 µg/mL of proteinase K and 3% hydrogen peroxide, sections were incubated with a labeling mixture for 1 h at 37 °C. Then 55 µL of anti-digoxigenin-peroxidase was deposited on the sections and incubated for 30 min. The reaction products were visualized by 3,3-diaminobenzidine substrate. Nuclei were counterstained with methyl green for 10 min. After washing with n-butanol, the sections were dehydrated, and mounted. Apoptotic index (%) corresponding to the number of labeled nuclei per 1 000 nuclei was calculated.
Statistical analysis
Experimental results were expressed as mean ±SE. Difference between the means was evaluated by the Mann-Whitney U test. P < 0.05 was considered statistically significant. The statistical analysis in this paper such as Kaplan-Meier analysis and Cox regression model was performed by using the software of StatView-5.0 Macintosh (Tokyo, Japan).
RESULTS
TP53INP1 was expressed in non-malignant gastric tissues and its expression was reduced in gastric cancer tissues
In the non-neoplastic gastric mucosa, TP53INP1 was mainly located in the cytoplasm of epithelial cells (Figures 1A and 1B). Some nuclei were also stained for TP53INP1 (Figure 2). Similar patterns were observed for intestinal metaplasia samples (Figure 1A). To determine if TP53INP1 is differentially expressed in gastric carcinomas or if it is stage-related, we did immunohistochemical analysis on 142 samples (76 cases of intestinal type, and 66 cases of diffuse type). All the cancer samples had accompanying non-malignant tissues, 98% of them were positive for TP53INP1 expression (Table 1) and thus could be used as internal control. In contrast, the expression of TP53INP1 protein was seen in 91 cases (64%), the other 51 samples (36%) were TP53INP1-negative (Figure 1B, arrow). Overall, TP53INP1 expression in gastric cancers was significantly lower both in cell cytoplasm and nucleus than in non-malignant gastric tissue (P < 0.0001, Table 1 and Figure 2). However, the expression of TP53INP1 was decreased in well-differentiated tubular adenocarcinoma (Figure 1C), and was markedly diminished in poorly differentiated-type cancer (Figure 1D).
Table 1 TP53INP1 expression in gastric cancer n (%).
Figure 2 Comparison of TP53INP1 expression (in cell cytoplasm and nucleus) between normal gastric mucosa and gastric cancer tissues (bP < 0.
0001).
We next examined whether TP53INP1 expression is associated or not with the development and progression of gastric carcinoma. The clinical details of the cohort of patients and the statistical analysis are listed in Table 2. Only two non significant associations were observed, i.e., age and gender. TP53INP1 negativity was associated with gastric body and antrum tumor location (P = 0.0193), with poorly differentiated adenocarcinoma (diffuse type) (P < 0.0001). With regard to the depth of invasion, the positive TP53INP1 expression rate was 100% in intramucosal tumors (5/5), 81.8% when mucosa was invaded (18/22), 76.5% in muscularis propria (26/34), 54.3% in subserosa (25/46), and 48.6% in serosa (17/35). These results showed that alteration of TP53INP1 expression was correlated to the staging of the tumors. The difference was statistically significant when T1 tumors were compared to the other stages (P = 0.0111, Table 2). In addition, TP53INP1 was significantly expressed in node-negative patients (P = 0.0037), and significantly associated with lymphatic invasion-negative patients (P = 0.0010). Taken together, these results indicated that loss of TP53INP1 expression was significantly associated with poorly differentiated histology, deep invasion, lymph node invasion, and metastasis.
Table 2 Correlation between TP53INP1 expression levels and clinicopathologic features in gastric cancer.
TP53INP1 modulates the cell cycle arrest and programmed cell death[3]. To investigate whether the modulation of TP53INP1 expression is associated with differences in apoptotic activity, TUNEL assays were done in all the 142 cases. TUNEL-positive nuclei were clearly seen in TP53INP1-positive (Figure 3A) and negative (Figure 3B) cancer lesions. As shown in Figure 3C, the apoptotic index in the TP53INP1-positive group (7.48 ± 2.66%) was significantly higher than that in the TP53INP1-negative group (4.16 ± 2.41%).
Figure 3 Apoptosis analysis in gastric cancer.
A and B: Representative patterns of TP53INP1-positive (A) and negative (B) carcinoma in TUNEL staining. Arrows indicate TUNEL-positive nuclei (original magnification ×20); C: Statistical analysis of apoptotic index in TP53INP1-positive and -negative cancer tissues (bP < 0.0001, positive vs negative).
TP53INP1 expression and prognosis
On univariate analysis, patient survival according to pathological stage was significantly different between TP53INP1-positive and TP53INP1-negative groups. Those patients with TP53INP1-positive expression had significantly better survival than those without TP53INP1 expression (P = 0.0006, Figure 4A). Survival for TP53INP1-positive patients with poorly differentiated adenocarcinoma was significantly longer than that of TP53INP1-negative patients (P = 0.0199, Figure 4B), whereas the survival of TP53INP1-positive patients in well or moderately differentiated adenocarcinoma was not significantly different from that of TP53INP1-negative patients (P = 0.1110, Figure 4C). Taken together, the results indicated that alteration of TP53INP1 expression was associated with a poor prognosis. Nevertheless, no prognostic value for TP53INP1 expression was evidenced from the multivariate analysis (Table 3). Histological type, apoptotic index, metastasis, and lymph node invasion were the most important independent prognostic factors, TP53INP1 could not be considered as an independent prognostic marker.
Table 3 Multivariate survival analysis using the Cox regression model.
Figure 4 TP53INP1 expression and patient survival by Kaplan-Meier analysis.
A: Survival curves for TP53INP1-positive and negative gastric cancers. The 60-mo survival rates were 74.7% and 54.9%, respectively. The difference between the values was highly significant (P = 0.0006). B: In well or moderately differentiated adenocarcinoma (intestinal-type), the survival of TP53INP1-positive patients was not significantly different from that of TP53INP1-negative group (P = 0.1110). C: In poorly differentiated adenocarcinoma (diffuse-type), the 60-mo patient survival rates were 62.1% and 45.9% for TP53INP1-positive and -negative gastric cancers, respectively. This difference was statistically significant (P = 0.0199).
P53 was not expressed in non-malignant gastric tissues but expressed in gastric cancer tissues
As shown in Table 1 and Figure 1E, the p53 protein was expressed in gastric cancer regions. whereas it was absent in non-malignant portions. The staining was nuclear. Cytoplasmic staining without nuclear staining was regarded as negative. The p53 positive rate in gastric cancer was significantly higher (47.9%) than that in non-malignant tissues (P < 0.05).
DISCUSSION
The development and progression of gastric cancer involve many types of genes that need to be activated or inactivated in order to promote malignancy. Gastric cancer is a heterogeneous pathology, classified into two general subtypes: intestinal (differentiated) and diffuse (undifferentiated)[17]. The intestinal type gastric carcinoma presents tumor suppressor gene alterations similar to colorectal tumors and distinct from diffuse type gastric cancer[18]. An accumulation of multiple genetic and epigenetic alterations of oncogenes, tumor suppressor genes, DNA repair genes, cell cycle regulators, cell adhesion molecules, and growth factor/receptor systems are involved during the multi-step conversion from normal epithelial cells to clinical gastric cancer[10-14]. TP53 gene alterations have been observed in both histological subtypes[19]. TP53INP1 is a tumor suppressor gene, located on the chromosome band 8q22[20]. Its expression is dependent on the activation of wide-type p53[3].
In this study, we have showed that TP53INP1 protein expression was significantly reduced in gastric cancer cells compared with non-cancerous adjacent tissues. We also reported that reduced TP53INP1 expression was associated with the diffuse cancer phenotype. Tomasini et al [2] have shown that TP53INP1 and HIPK2 are partners in regulating p53 activity. It is increasingly evident that methylation of CpG islands in the promoter of specific tumor suppressor genes, such as p16, is associated with their silencing in human gastric cancer[21]. The 5′-upstream region of TP53INP1 contains a CpG island. The sequences from nucleotide -792, the region on exon 1 and part of the first intron, to nucleotide +839 have the highest content of CpG dinucleotides. However, there was no mutation of TP53INP1 gene in pancreatic carcinoma (unpublished data). In addition, the reduced TP53INP1 expression in gastric cancer especially in the diffuse type. may relate to the wide-type p53 inactivation in gastric cancer.
We showed that p53 was expressed in 68 cases of gastric cancer, whereas it was not present in normal gastric epithelial cells. We observed that in non-gastric cancer regions the expression of TP53INP1 was opposite to that of p53. The expression of TP53INP1 was dependent on the wild type p53 since the wild-type p53 protein is biologically unstable and has a shorter half-life than mutant p53 protein[24]. This characteristic of wide-type p53 protein does not allow it to be detected by immunohistochemical methods, but mutant p53 can be detected by immunostaining. Our data was similar to Carvalho et al [22] who showed that there was no difference of the p53 expression between the intestinal type and the diffuse type in gastric cancer. Whereas Lin et al [23] showed the expression of p53 in the intestinal type was more frequent than that in the diffuse type. The precise mechanisms of TP53INP1 suppression in gastric cancer need further research.
The mechanism of the suppression of these genes in poorly differentiated gastric carcinoma is not clear. Many studies suggested that most tumor suppressor genes play a role in mediating cell cycle arrest in the G1 phase following DNA damage and also function in the removal of damaged cells by initiating apoptosis in certain physiological situations[10]. TP53INP1 and HIPK2 are partners in regulating p53 activity[2]. Overexpression of TP53INP1 induces G1 cell cycle arrest. TP53INP1 expression was significantly decreased in advanced gastric cancers. These results suggest that decrease of TP53INP1 expression might play an important role in the progression of gastric cancer. Assessment of TP53INP1 expression level may serve as a novel biomarker for predicting the malignant grade of cancers, like another marker for poor prognosis genes[25,26].
Lymphatic involvement is thought to be important as an initial step of lymph node metastasis[27]. Our study showed that TP53INP1 expression was significantly reduced in lymphatic invasion-positive groups. TP53INP1 expression decreased in node-positive. Taken together, these results suggested that loss of TP53INP1 expression is associated with lymph node metastasis of gastric cancer.
Deregulation of genes involved in cell cycle and cell signaling pathways has been described and classified as early events for cyclin D1 and p16 genes or late events for p53, DPC4 and BRCA2 genes in the progression model studies[28]. TP53INP1 is a pro-apoptotic gene strongly activated during cell stress. Overexpression of TP53INP1 is related to G1 cell cycle arrest and induces p53-mediated cell death[3]. In the present study, we showed that the apoptotic index in TP53INP1-positive lesions was higher than that in TP53INP1-negative lesions in gastric cancer tissues as detected by TUNEL assay, indicating that TP53INP1 is related to apoptosis and tumor aggre-ssiveness in gastric carcinoma.
TP53INP is an acute gene that is induced by various stresses such as UV, heat shock, etc.[1]. Up to now, we have known little function of it. We for the first time revealed the impact of the TP53INP1 on the survival in gastric cancer. We showed that the survival rate of the TP53INP1-positive cases was longer than that of the TP53INP1-negative cases, especially in the diffuse type gastric cancer.
In conclusion, the present study has showed that the reduction of TP53INP1 expression might play roles in gastric carcinogenesis and tumor aggressiveness. Analysis of the TP53INP1 may be useful to evaluate the malignant grade of gastric cancer.
Footnotes
S- Editor Guo SY L- Editor Elsevier HK E- Editor Cao L
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