Skip to main content
NCBI home page
Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2007 Sep 18;134(4):481–488. doi: 10.1007/s00432-007-0310-1

Aberrant Pim-3 expression is involved in gastric adenoma–adenocarcinoma sequence and cancer progression

Hua-Chuan Zheng 1,5, Koichi Tsuneyama 1,2,✉, Hiroyuki Takahashi 1, Shigeharu Miwa 3, Toshiro Sugiyama 3, Boryana Konstantinova Popivanova 4, Chifumi Fujii 4, Kazuhiro Nomoto 1, Naofumi Mukaida 4, Yasuo Takano 1
PMCID: PMC12161629  PMID: 17876606

Abstract

Purpose

Pim-3, a member of the proto-oncogene Pim family with serine/threonine kinase activity was aberrantly expressed in cancerous lesions of endoderm-derived organs such as liver, pancreas, and colon. The aim of this study was to clarify the role of Pim-3 expression in the tumorigenesis and the development of gastric carcinomas.

Methods

Pim-3 expression was immunohistochemically examined on the tissue microarrays containing primary (n = 285) and metastastic (n = 37) sites of gastric carcinomas, in comparison with adenoma (n = 48) and non-cancerous mucosa (n = 84). It was also compared with the clinicopathological parameters of gastric carcinomas.

Results

Pim-3 expression was enhanced in adenoma (64.6%) and metastasis sites of gastric carcinoma (73.0%), to a lesser degree in primary sites of gastric carcinoma (39.3%) when compared to non-cancerous mucosa (13.1%, p < 0.0001). Pim-3 expression levels were higher in intestinal-type than diffuse-type gastric carcinoma (p = 0.018). Pim-3 expression was closely correlated with sex (p = 0.047), lymphatic (p = 0.019) and venous invasion (p = 0.014). Pim-3 expression was correlated significantly with vascular endothelial growth factor (VEGF, p = 0.009) and extracellular matrix metalloproteinase inducer (EMMPRIN, p = 0.032), both of which are presumed to be involved in neovascularization, a crucial step for metastasis. On the contrary, phosphatase and tensin homology deleted from human chromosome 10 (Pten) negative gastric carcinomas exhibited higher Pim-3 expression than Pten positive ones (p = 0.042). There was no relationship between Pim-3 expression and MVD in gastric carcinomas (p = 0.715). Furthermore, patients with Pim-3 positive gastric cancer, showed a lower cumulative survival rate than those with Pim-3 negative gastric cancer (p = 0.014) and Pim-3 positive was also identified as an independent prognostic factor for gastric carcinoma patients (p = 0.006).

Conclusions

Aberrant Pim-3 expression was involved in gastric adenoma–adenocarcinoma sequence and subsequent invasion and metastasis process in gastric cancer. Moreover, Pim-3 may be employed to predict the prognosis of gastric cancer patients. Distinct Pim-3 expression underlies the molecular mechanisms for the differentiation of intestinal-type and diffuse-type carcinomas.

Keywords: Angiogenesis, Gastric carcinoma, Invasion, Metastasis, Pim-3, Prognosis

Introduction

KID-1 was identified as depolarization-induced gene in a rat pheochromocytoma cell line PC121 and was presumed to be involved in the neuronal cell functions including consolidation of long-term potentiation (Feldman et al. 1998). Concomitantly, based on a high sequence similarity of KID-1 with Pim-1 and Pim-2, members of a proto-oncogene Pim family, KID-1 was renamed as Pim-3 (Konietzko et al. 1999). Deneen et al. (2003) demonstrated that Pim-3 gene transcription was enhanced in EWS/ETS-induced malignant transformation of NIH 3T3 cells, suggesting the potential involvement of Pim-3 in tumorigenesis. Supporting this notion, we previously observed that the ablation of Pim-3 expression induced the apoptosis of human hepatocellular, pancreas, and colon carcinoma cell lines (Fujii et al. 2005; Li et al. 2006; Popivanova et al. 2007). Moreover, we have revealed that Pim-3 can inactivate a potent pro-apoptotic molecule, which is bad in human pancreas and colon carcinoma cell lines, by phosphorylating its Ser112, and thereby preventing apoptosis (Li et al. 2006; Popivanova et al. 2007). Furthermore, Pim-3 is selectively expressed in pre-cancerous to cancerous lesions but not normal tissues from endoderm-derived organs such as liver, pancreas, and colon (Fuji et al. 2005; Li et al. 2006; Popivanova et al. 2007). Thus, it is tempting to speculate that Pim-3 expression is aberrantly expressed in pre-cancerous to cancerous lesions of another endoderm-derived organ, stomach, which does not express Pim-3 in a normal state.

Aberrant expression of vascular endothelial growth factor (VEGF) is crucially involved in tumor neovasculaziation, an indispensable step for tumor growth (Zheng et al. 2006a). Prostate-specific antigen reduced the in vivo growth of a human prostate cancer cell line PC-3M, in nude mice, together with down-regulation of VEGF and Pim-1 expression (Bindukumar et al. 2005). Helicobacter pylori obtained from gastric cancer and duodenal ulcer patients also induced the expression of both Pim-1 and VEGF in human gastric epithelial cells (Chang et al. 2006). Furthermore, Pim-1 was identified as a target gene in vasculogenesis mediated by VEGF receptor-2 (Zippo et al. 2004). These observations suggest the potential interaction between VEGF and Pim-1. Even at the amino acid level, human Pim-3 shows a high sequence similarity with human Pim-1 (57.1%). It is tempting to speculate that Pim-3 may also interact with VEGF.

Tumorigenesis and progression of gastric carcinoma are multistage processes accompanied with changes in gene expression (Zheng et al. 2006b). Phosphatase and tensin homology deleted from human chromosome 10 (Pten) gene showed frequent deletion and down-regulated expression in human gastric cancer (Chang et al. 1999; Zheng et al. 2003). Pten negatively regulates the activity of Akt and therefore, Pten deletion resulted in Akt overexpression in colon and pancreas cancers (Roy et al. 2002; Li et al. 2004), similar to that by Pim-3. Concomitantly, Pten deletion is associated with poor prognosis of gastric cancer patients, together with enhanced angiogenesis and matrix metalloproteinase (MMP) expression (Zheng et al. 2007a; 2006a). Moreover, we recently observed that extracellular MMP inducer (EMMPRIN) expression was enhanced in gastric carcinoma together with enhanced expression of VEGF and MMPs (Zheng et al. 2006a, c).

The aforementioned observations prompted us to assume that Pim-3 expression might be enhanced in pre-cancerous and cancerous lesions of stomach. Hence, in the present study, we investigated Pim-3 expression in gastric non-cancerous mucosa, adenoma, and adenocarcinoma at both primary and metastatic sites, with reference to the expression of Pten, VEGF, and EMMPRIN, which are presumed to be involved in gastric carcinogenesis.

Materials and methods

Subjects

Primary gastric carcinoma (n = 285), metastatic carcinoma (liver metastases, n = 2; lymph node metastases, n = 35), adenoma (n = 41), adenoma adjacent to carcinoma (n = 7), and non-cancerous mucosa (n = 84) were collected during surgical operation or endoscopy submucosa dissection (ESD) in our affiliated hospital and its related institutes between 1993 and 2005. None of the patients underwent chemotherapy or radiotherapy before the resection. They all provided consent for the use of tumor tissue for clinical research and our University Ethical Committee approved the research protocol. We followed up the patients by consulting their case documents and through telephone.

Cell lines and culture

Gastric carcinoma cell lines, HGC-27 (undifferentiated adenocarcinoma), MKN28 (well-differentiated adenocarcinoma), MNK45 (poorly differentiated adenocarcinoma), KATO-III (poorly-differentiated adenocarcinoma), AGS (moderately differentiated adenocarcinoma) come from Japanese Physical and Chemical Institute. They were maintained in RPMI 1640 (MKN28, MKN45 and KATO), MEM (HGC-27) and Ham F12 (AGS) media supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin, in a humidified atmosphere of 5% CO2 at 37°C. Total protein was extracted from cells using cell disruption buffer according to the PARIS manual (Arctiris Bioscience, USA). All cells were collected by centrifugation, rinsed with PBS, fixed by 10% formalin , and then embedded in paraffin.

Pathological analysis and tissue microarray (TMA)

Tissues were fixed in 4% neutralized formaldehyde, embedded in paraffin and incised into 4 μm thick sections. These sections were stained by hematoxylin and eosin (HE) to confirm their histological diagnosis and other microscopic characteristics including the depth of invasion, lymphatic and venous invasion. The staging for each gastric carcinoma was determined according to the Union Internationale Contre le Cancer (UICC) system for the extent of tumor spread (Sobin and Wittekind 2002). Histological appearance of tumors was expressed according to Lauren’s classification (Zheng et al. 2007b). From the selected tumor cases, representative areas of solid tumors and non-cancerous mucosa were further identified in HE stained sections. Then, a tissue core with a diameter of 2 mm was punched out from each donor block and transferred to a recipient block with a maximum of 48 cores using a tissue microarrayer (AZUMAYA KIN-1, Tokyo, Japan). Four micrometer thick sections were consecutively incised from the recipient block and transferred to poly-lysine-coated glass slides. HE staining was performed on TMA for the confirmation of tumor tissues (Fig. 1a).

Fig. 1.

Fig. 1

Open in a new tab

HE staining and immunostaining on TMA. a HE-stained TMA section (original magnification,(×40), b, c immunohistochemical analysis using anti-Pim-3 was performed non-cancerous mucosa d adenoma, e, f The primary site of intestinal-type carcinoma, g diffuse-type carcinoma, h lymph node metastasis site, as described in “Materials and methods”. Gastric carcinoma tissue was immunostained with i anti-Pten, j anti-EMMPRIN, k anti-VEGF antibodies, as described in “Materials and methods” (original magnification, ×400). l CD34 was localized in the membrane and cytoplasm of vascular epithelial cell to label MVD (original magnification ×400)

Immunohistochemical analysis

Consecutive sections were deparaffinized with xylene, dehydrated with alcohol, and subjected to antigen retrieval by irradiating in target retrieval solution (TRS, DAKO, Carpinteria, CA 93013, USA) for 5 min with a microwave oven (Oriental rotor Lmt. Co., Tokyo, Japan). Five percent bovine serum albumin was then applied for 1 min to prevent non-specific binding. The sections were incubated with either rabbit anti-Pim-3 (Popivanova et al. 2007, 1:1000), mouse anti-Pten (Novocastra, New Castle upon Tyne NE 1282 W, UK; 1:150), mouse anti-EMMPRIN (Novocastra, UK; 1:50), rabbit anti-VEGF antibodies (LABVISION, Fremont CA 94530, USA; ready to use), or mouse anti-CD34 (DAKO, USA; 1:100) for 15 min. The slides were treated with either anti-mouse or anti-rabbit Envison-PO antibodies (DAKO, USA) for 15 min. All incubations were performed in a microwave oven to allow intermittent irradiation as described previously Kumada et al (2004). After each treatment, the slides were washed with TBST (10 mM Tris–HCl, 150 mM NaCl, 0.1% Tween 20) three times for 1 min. Binding sites were visualized with 3,3′-diaminobenzidine (DAB). After counterstain with Mayer’s hematoxylin, the sections were dehydrated, cleared and mounted. Omission of the primary antibody was used as a negative control. One hundred cells were randomly selected and counted on five representative fields of each section blindly by three independent observers (K. Tsuneyama, Y. Takano, H-C. Zheng). For Pim-3 evaluation, the positive percentage of counted cells was graded semi-quantitatively according to a five-tier scoring system: negative (−), 0%; very weakly positive (±), 1–5%; weakly positive (+), 6–25%; moderately positive (++), 26–50%; and strongly positive (+++), 51–100%. As for Pten, VEGF , and EMPPRIN, more than 5% of positively stained cells were regarded as be positive (+).

Microvessel density counting

CD34 expression in the cytoplasm and membrane of vascular epithelial cells (Fig. 1l) was selected for the microvessel density counting although it was occasionally localized in tumor cells and fibroblasts. A modified Weidner’s method was used to calculate the microvessel density of gastric carcinoma after anti-CD34 immunostaining (Weidner 1995). In brief, observers selected five representative areas and counted individual microvessels on a ×400 field (0.1885 mm2/field) after the area of highest neovascularization was identified. Brown staining endothelial cells or endothelial cell clusters, clearly separated from the adjacent microvessel, tumor cells, and other connective tissue elements were considered as a single, countable microvessels. Three investigators performed the counts independently.

Western blot

One hundred microgram denatured protein was separated on an SDS–polycrylamide gel (10% acrylamide) and transferred to Hybond membrane (Amersham, Germany), which was then blocked overnight in 5% milk in TBST. For immunoblotting, the membrane was incubated for 1 h with the antibody against Pim-3 (1:1,000). Then it was rinsed by TTBS and incubated with anti-rabbit IgG conjugated to horseradish peroxidase (DAKO, USA, 1:1000) for 1 h. Bands were visualized on X-ray film (Fujifilm, Japan) using Amersham ECL-Plus detection reagents (Amersham, Germany). After that, membrane was washed with WB stripping solution of pH 2–3 (Nacalai, Japan) for 1 h and treated as described above, except mouse β-actin antibody (Sigma, USA, 1:5,000) as internal control.

Statistical analysis

Statistical evaluation was performed using Spearman correlation test to analyze the rank data and using Mann–Whitney U test to differentiate the means of different groups. Kaplan–Meier survival plots were generated and comparisons between survival curves were made with the log-rank statistic. The Cox proportional hazards model was employed for multivariate analysis, p < 0.05 was considered as statistically significant.

Results

Pim-3 expression in gastric non-cancerous mucosa, adenoma, carcinoma and its metastasis

Non-cancerous epithelial cells were negative (Fig. 1b) or weakly positive for Pim-3 (Fig. 1c). Pim-3 was present in cytoplasm of non-cancerous epithelium, adenoma, and adenocarcinoma while the distribution changed depending on the types of the lesions. Pim-3 was mainly distributed to a cavity-oriented perinuclear area with a dot-like pattern in gastric non-cancerous epithelium, adenoma and intestinal-type carcinomas (Fig. 1c–f), whereas it was detected diffusely in the cytoplasm of the diffuse-type carcinomas (Fig. 1g). There was no Pim-3 expression detected in five kinds of gastric cell lines by immunohistochemistry or western blot (data not shown). In a tissue section with the mixture of adenoma and adenocarcinoma, Pim-3 expression was stronger in gastric adenoma, compared to that of adjacent non-cancerous mucosa (Fig. 1b, c), but became weaker in adenocarcinoma portion (Fig. 2). In line with these observations, the incidence and staining intensities of Pim-3 expression were higher in gastric adenoma, than in carcinoma, while few cells in non-cancerous mucosa were positive for Pim-3 (p < 0.0001, Fig. 1h; Table 1). It is interesting that the cancer cells in metastatic foci exhibited higher positive reactions than those in the primary site (p < 0.0001, Table 1). Pim-3 expression in a primary site of gastric cancer was positively correlated with patient sex (p = 0.047), lymphatic (p = 0.019), and venous invasion (p = 0.014), and a higher Pim-3 expression was observed in intestinal-type gastric carcinoma, compared with diffuse-type one (p = 0.018, Table 2). In contrast, patient age (p = 0.159), tumor size (p = 0.941), depth of invasion into gastric mucosa (p = 0.729), lymph node metastasis (p = 0.660), and tumor staging (p = 0.754) did not have any significant effects on Pim-3 expression (Table 2). Because of the VEGF, Pten, and EMMPRIN were presumed to be involved in gastric carcinogenesis, we compared Pim-3 expression with the expression of these molecules in gastric carcinomas. Pim-3 expression was positively correlated with those of VEGF (p = 0.009) and EMMPRIN (p = 0.032), but negatively with Pten expression (p = 0.042, Fig. 1i–k; Table 2). However, there was no significant relationship between Pim-3 expression and microvessel density in gastric carcinomas (p = 0.715, Fig. 3).

Fig. 2.

Fig. 2

Open in a new tab

HE staining and immunostaining on gastric neoplasia from ESD. Serial sections from an ESD sample were stained with HE (left panels) or immunostained with anti-Pim-3 antibodies (right panels). The boxes in the upper panels indicate the sites observed under a higher magnification. 1 and 2 represent adenoma and adenocarcinoma in situ, respectively (original magnification: upper panels, ×100; lower panels, ×400)

Table 1.

Pim-3 expression in gastric non-cancerous mucosa adenoma, carcinoma , and its metastases

Groups N Pim-3 expression
− ± + ++ +++ PR (%)
Non-cancerous mucosa 84 73 7 4 0 0 13.1
Adenoma 48 17 7 11 7 6 64.6a
Primary Carcinoma 285 173 49 30 22 11 39.3b
Metastastic carcinoma 37 10 3 5 8 11 73.0c
Open in a new tab

Spearman’s correlation test: acompared with the non-cancerous mucosa, p < 0.0001 or carcinoma, p < 0.0001; bcompared with the non-cancerous mucosa, p < 0.0001; ccompared with the primary carcinoma; PR positive rate

Table 2.

Relationship between Pim-3 expression and clinicopathological parameters of gastric carcinomas

Clinicopathological features N Pim-3 expression
− ± + ++ +++ PR (%) p value
Sex 0.047
  Female 67 47 10 6 3 1 29.9
  Male 201 123 39 14 17 8 38.8
Age 0.159
  <60 76 54 14 4 4 0 28.9
  ≥60 192 116 35 16 16 9 39.6
Tumor size (cm) 0.941
  <4 142 90 26 12 9 5 36.6
  ≥4 126 80 23 8 11 4 36.5
Depth of invasion 0.729
  Tis–T1 145 92 30 10 7 6 35.2
  T2–T4 123 78 19 10 13 3 36.6
Lymphatic invasion 0.019
  − 175 120 28 12 9 6 31.4
  + 93 50 21 8 11 3 43.0
Venous invasion 0.014
  − 240 157 44 18 15 6 34.6
  + 28 13 5 2 5 3 53.6
Lymph node metastasis 0.660
  − 162 104 31 12 8 7 35.8
  + 106 66 18 8 12 2 37.7
UICC staging 0.754
  O/I 175 112 33 12 11 7 36.0
  II, III/IV 93 58 16 8 9 2 37.6
Lauren’s classification 0.018
  Intestinal-type 146 82 34 13 12 5 43.8
  Diffuse-type 122 88 15 7 8 4 27.9
Pten expression 0.042
  − 90 56 14 7 11 2 37.8
  + 178 114 35 13 9 7 36.2
VEGF expression 0.009
  − 84 62 17 4 1 0 26.2
  + 184 108 32 16 19 9 41.3
EMMPRIN expression 0.032
  − 119 85 21 7 6 0 28.6
  + 149 85 28 13 14 9 43.0
Open in a new tab

Spearman’s correlation test: T is carcinoma in situ, T 1 lamina propria and submucosa, T 2 muscularis propria and subserosa, T 3 exposure to serosa, T 4 invasion into serosa, PR positive rate

Fig. 3.

Fig. 3

Open in a new tab

Relationship between Pim-3 expression and microvessel density in gastric carcinomas

Univariate and multivariate survival analysis

Follow-up information was available on 255 patients with gastric carcinoma for a period ranging from 0.2 months to 12.2 years (median = 68.6 months). Univariate analysis using Kaplan–Meier method demonstrated that cumulative rate of the patients with negative Pim-3 expression was significantly higher than those with positive Pim-3 expression even stratified by the depth of invasion (Fig. 4, p = 0.014). Moreover, multivariate analysis using the Cox’s proportional hazard model indicated that the depth of invasion (p = 0.005), lymphatic (p = 0.001), and venous invasion (p = 0.017), peritoneal dissemination (p = 0.013), and Pim-3 expression (p = 0.006) were all independent prognostic factors for the gastric carcinoma patients (Table 3).

Fig. 4.

Fig. 4

Open in a new tab

Effects of Pim-3 expression on survival rates among gastric carcinoma patients. Kaplan–Meier curves for cumulative survival rate were obtained on patients with gastric carcinomas, stratified according to Pim-3 expression

Table 3.

Multivariate analyses of clinical variables for gastric carcinomas

Clinicopathological parameters Relative risk (95% CI) p value
Tumor size (≥4 cm) 1.468 (0.832–2.588) 0.166
Depth of invasion (Tis, 1/T2, 3) 3.675 (1.645–8.210) 0.005
Lymphatic invasion (−/+) 3.304 (1.754–5.246) 0.001
Venous invasion (−/+) 1.722 (1.047–2.830) 0.017
Lymph node metastasis (−/+) 1.869 (0.714–4.891) 0.148
Peritoneal dissemination (−/+) 4.963 (2.026–12.157) 0.013
Liver metastasis (−/+) 1.631 (0.623–4.273) 0.230
UICC staging (O–I/II–IV) 0.574 (0.218–1.512) 0.266
Differentiation (Intestinal/diffuse) 1.124 (0.716–1.762) 0.419
Pim-3 expression (−/±/+/++/+++) 1.227 (1.029–1.464) 0.006
Open in a new tab

CI confidence interval

Discussion

We have previously observed that a proto-oncogene Pim-3 with serine/threonine kinase activity was selectively expressed in pre-cancerous and cancerous lesions of endoderm-derived organs including liver, pancreas, and colon (Fuji et al. 2005; Li et al. 2006; Popivanova et al. 2007). Moreover, Pim-3 can inactivate a pro-apoptotic molecule, which is bad on phosphorylation at its serine residue, and thereby prevent apoptosis (Li et al. 2006; Popivanova et al. 2007). These observations prompted us to speculate that the Pim-3 expression is also enhanced in pre-cancerous and cancerous lesions in another endoderm-derived organ, stomach. Indeed, Pim-3 expression was enhanced with a high frequency in adenoma and to a lesser degree, adenocarcinoma tissues, compared with non-cancerous normal mucosa, as similar to that observed on colonic adenoma and adenocarcinoma tissues (Popivanova et al. 2007). Thus, Pim-3 expression may be a good biomarker for gastrointestinal adenoma, in general. Actually, the adenoma is incorporated with gastric well-differentiated carcinoma when it grows bigger and de novo carcinogenesis is well understood, especially in diffuse-type gastric carcinomas. Higher Pim-3 expression in adenoma and intestinal-type carcinoma indicated that aberrant Pim-3 expression might play an important role in intestinal-type carcinogenesis, but less in de novo carcinogenic pathway.

Pim-3 expression was detected in gastric cancer tissues with a frequency of <40%, which is lower than adenoma tissues. There was no Pim-3 expression detected in five gastric carcinoma cell lines by immunohistochemistry and even western blot. Down-regulated Pim-3 expression was suggested to involve in the gastric adenoma–adenocarcinoma sequence. We observed that Pim-3 expression was similarly enhanced with a higher frequency in pre-cancerous lesions than cancerous lesions in liver and colon (Fuji et al. 2005; Popivanova et al. 2007). One main function of Pim-3 seems to be the prevention of apoptosis. It is interesting to know that, a higher frequency of apoptosis has been observed in gastric dysplasia than gastric carcinoma, and an anti-apoptotic molecule Bcl-2, was similarly up-regulated in pre-malignant lesions but down-regulated after malignant transformation (Xia and Talley 2001). Thus, Pim-3 may be aberrantly expressed in adenoma, in order to counteract apoptosis of dysplastic cells present in adenoma tissues. This assumption may be further supported by our present observations that Pim-3 was expressed with a higher incidence in intestinal-type gastric cancer, which is presumed to arise from preceding dysplastic lesions (Faraji and Frank 2002), than diffuse-type one, which evolve without any precedent dysplastic changes. It is also demonstrated that distinct Pim-3 expression underlies the molecular mechanisms for the differentiation of intestinal-type and diffuse-type carcinomas.

Subtractive suppression hybridization revealed that Pim-3 expression was augmented in metastatic 5–8F nasopharyngeal carcinoma cell line, compared with non-metastatic 6–10B (Yang et al. 2005). Also in the primary site of gastric carcinoma, we observed a positive correlation of Pim-3 expression with the lymphatic and venous invasion of cancer cells, which is the first step of metastasis. Moreover, metastatic sites exhibited Pim-3 expression with a higher incidence than primary sites. Moreover, our analysis has revealed that Pim-3 was an independent prognostic factor for the patients with gastric carcinoma and patients with Pim-3 positive gastric cancer showed a lower cumulative survival rate than those with Pim-3 negative gastric cancer. Therefore, Pim-3 may be a good indicator to predict the worst prognosis of patients with gastric carcinoma. Although TNM staging, lymph nodal involvement , and hepatic metastasis were reported to be the most significant prognostic factors for gastric carcinomas (Celen et al. 2007; Juvan et al. 2007; Shirabe et al. 2003), significant results were not observed in the Cox’s proportional hazard analysis of the present study. The discrepancy might be attributable to clinicopathological characteristics of the subjects and grouping methods in multivariate analysis.

Pten can suppress angiogenesis by reducing expression of VEGF and MMPs, and both of them are crucially involved in metastasis process (Zheng et al. 2006a). EMMPRIN, a glycosylated cell surface transmembrane protein can augment VEGF and MMP expression in the neighboring fibroblasts or epithelial cells in a paracrine manner (Zheng et al. 2006c). Pten negatively modulates the activity of Akt, a serine/threonine kinase that phosphorylates similar sets of substrates as Pim kinases including Pim-3 (Amaravadi and Thompson 2005) and Pten gene deletion resulted in Akt overexpression in various types of cancer including gastric cancer (Pedrero et al. 2005, Knobbe and Reifenberger 2003; Tang et al. 2006; Oki et al. 2005). Thus, we investigated the expression of Pten, VEGF, and EMMPRIN in parallel. Pten negative gastric carcinoma exhibited higher levels of Pim-3 expression and Pim-3 expression was correlated with VEGF and EMMPRIN expression. Thus, Pim-3 may induce angiogenesis in gastric cancer tissue, in combination with overexpression of VEGF and EMMPRIN, and Pten loss. However, no close association between Pim-3 expression and MVD was found in the this study, possibly due to the low positive rate of Pim-3 expression or the weak effects of Pim-3 on angiogenesis in gastric carcinomas. Elucidation on the interrelationship between Pim-3 and these molecules, at molecular levels will shed a novel light on the molecular mechanisms underlying carcinogenesis and subsequent invasion and metastasis process in stomach.

Acknowledgments

We particularly thank Kanako Yasuyoshi, Tokimasa Kumada and Hideki Hatta for their technical help and Yukari Inoue for her secretarial assistance. This work was partially supported by the Japanese Ministry of Education, Science, Sports and Culture, Grant-in-Aid for Scientific Research 14770072, 18590324 and Japanese Smoking Foundation.

References

  1. Amaravadi R, Thompson CB (2005) The survival kinases Akt and Pim as potential pharmacological targets. J Clin Invest 115:2618–2624 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bindukumar B, Schwartz SA, Nair MP, Aalinkeel R, Kawinski E, Chadha KC (2005) Prostate-specific antigen modulates the expression of genes involved in prostate tumor growth. Neoplasia 7:241–252 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Celen O, Yildirim E, Berberoglu U (2007) Prognostic impact of positive lymph node ratio in gastric carcinoma. J Surg Oncol 96:95–101 [DOI] [PubMed] [Google Scholar]
  4. Chang JG, Chen YJ, Perng LI, Wang NM, Kao MC, Yang TY, Chang CP, Tsai CH (1999) Mutation analysis of the PTEN/MMAC1 gene in cancers of the digestive tract. Eur J Cancer 35:647–651 [DOI] [PubMed] [Google Scholar]
  5. Chang YT, Wu MS, Chang YJ, Chen CC, Lin YS, Hsieh T, Yang PC, Lin JT (2006) Distinct gene expression profiles in gastric epithelial cells induced by different clinical isolates of Helicobacter pylori-implication of bacteria and host interaction in gastric carcinogenesis. Hepatogastroenterology 53:484–490 [PubMed] [Google Scholar]
  6. Deneen B, Welford SM, Ho T, Hernandez F, Kurland I, Denny CT (2003) PIM3 proto-oncogene kinase is a common transcriptional target of divergent EWS/ETS oncoproteins. Mol Cell Biol 23:3897–3908 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Faraji EI, Frank BB (2002) Multifocal atrophic gastritis and gastric carcinoma. Gastroenterol Clin North Am 31:499–516 [DOI] [PubMed] [Google Scholar]
  8. Feldman JD, Vician L, Crispino M, Tocco G, Marcheselli VL, Bazan NG, Baudry M, Herschman HR (1998) KID-1, a protein kinase induced by depolarization in brain. J Biol Chem 273:16535–16543 [DOI] [PubMed] [Google Scholar]
  9. Fujii C, Nakamoto Y, Lu P, Tsuneyama K, Popivanova BK, Kaneko S, Mukaida N (2005) Aberrant expression of serine/threonine kinase Pim-3 in hepatocellular carcinoma development and its role in the proliferation of human hepatoma cell lines. Int J Cancer 114:209–218 [DOI] [PubMed] [Google Scholar]
  10. Juvan R, Hudler P, Gazvoda B, Repse S, Bracko M, Komel R (2007) Significance of genetic abnormalities of p53 protein in Slovenian patients with gastric carcinoma. Croat Med J 48:207–217 [PMC free article] [PubMed] [Google Scholar]
  11. Knobbe CB, Reifenberger G (2003) Genetic alterations and aberrant expression of genes related to the phosphatidyl-inositol-3′-kinase/protein kinase B (Akt) signal transduction pathway in glioblastomas. Brain Pathol 13:507–518 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Konietzko U, Kauselmann G, Scafidi J, Staubli U, Mikkers H, Berns A, Schweizer M, Waltereit R, Kuhl D (1999) Pim kinase expression is induced by LTP stimulation and required for the consolidation of enduring LTP. EMBO J 18:3359–3369 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kumada T, Tsuneyama K, Hatta H, Ishizawa S, Takano Y (2004) Improved 1–h rapid immunostaining method using intermittent microwave irradiation: practicability based on 5 years application in Toyama Medical and Pharmaceutical University Hospital. Mod Pathol 17:1141–1149 [DOI] [PubMed] [Google Scholar]
  14. Li D, Xie K, Wolff R, Abbruzzese JL (2004) Pancreatic cancer. Lancet 363:1049–1057 [DOI] [PubMed] [Google Scholar]
  15. Li YY, Popivanova BK, Nagai Y, Ishikura H, Fujii C, Mukaida N (2006) Pim-3, a proto-oncogene with serine/threonine kinase activity, is aberrantly expressed in human pancreatic cancer and phosphorylates bad to block bad-mediated apoptosis in human pancreatic cancer cell lines. Cancer Res 66:6741–6747 [DOI] [PubMed] [Google Scholar]
  16. Oki E, Baba H, Tokunaga E, Nakamura T, Ueda N, Futatsugi M, Mashino K, Yamamoto M, Ikebe M, Kakeji Y, Maehara Y (2005) Akt phosphorylation associates with LOH of PTEN and leads to chemoresistance for gastric cancer. Int J Cancer 117:376–380 [DOI] [PubMed] [Google Scholar]
  17. Pedrero JM, Carracedo DG, Pinto CM, Zapatero AH, Rodrigo JP, Nieto CS, Gonzalez MV (2005) Frequent genetic and biochemical alterations of the PI 3-K/AKT/PTEN pathway in head and neck squamous cell carcinoma. Int J Cancer 114:242–248 [DOI] [PubMed] [Google Scholar]
  18. Popivanova BK, Li YY, Zheng H, Fuji C, Tsuneyama K, Mukaida N (2007) A proto-oncogene, Pim-3 with serine/threonin kinase activity, is aberrantly expressed in human colon cancer cells, accompanied with Bad phosphorylation. Cancer Sci 98:321–328 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Roy HK, Olusola BF, Clemens DL, Karolski WJ, Ratashak A, Lynch HT, Smyrk TC (2002) AKT proto-oncogene overexpression is an early event during sporadic colon carcinogenesis. Carcinogenesis 23:201–205 [DOI] [PubMed] [Google Scholar]
  20. Shirabe K, Shimada M, Matsumata T, Higashi H, Yakeishi Y, Wakiyama S, Ikeda Y, Ezaki T, Fukuzawa S, Takenaka K, Kishikawa K, Ikeda T, Taguchi K, Maehara Y, Sugimachi K (2003) Analysis of the prognostic factors for liver metastasis of gastric cancer after hepatic resection: a multi-institutional study of the indications for resection. Hepatogastroenterology 50:1560–1563 [PubMed] [Google Scholar]
  21. Sobin LH, Wittekind CH (2002) TNM Classification of Malignant Tumors, 6th edn. Wiley, New Jersey [Google Scholar]
  22. Tang JM, He QY, Guo RX, Chang XJ (2006) Phosphorylated Akt overexpression and loss of PTEN expression in non-small cell lung cancer confers poor prognosis. Lung Cancer 51:181–191 [DOI] [PubMed] [Google Scholar]
  23. Weidner N (1995) Current pathologic methods for measuring intratumoral microvessel density within breast carcinoma and other solid tumors. Breast Cancer Res Treat 36:169–180 [DOI] [PubMed] [Google Scholar]
  24. Xia HH, Talley NJ (2001) Apoptosis in gastric epithelium induced by Helicobacter pylori infection: implications in gastric carcinogenesis. Am J Gastroenterol 96:16–26 [DOI] [PubMed] [Google Scholar]
  25. Yang XY, Ren CP, Wang L, Li H, Jiang CJ, Zhang HB, Zhao M, Yao KT (2005) Identification of differentially expressed genes in metastatic and non-metastatic nasopharyngeal carcinoma cells by suppression subtractive hybridization. Cell Oncol 27:215–223 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Zheng HC, Sun JM, Li XH, Yang XF, Zhang YC, Xin Y (2003) Role of PTEN and MMP-7 expression in growth, invasion, metastasis and angiogenesis of gastric carcinoma. Pathol Int 53:659–666 [DOI] [PubMed] [Google Scholar]
  27. Zheng H, Takahashi H, Murai Y, Cui Z, Nomoto K, Niwa H, Tsuneyama K, Takano Y (2006a) Expressions of MMP-2, MMP-9, and VEGF were closely linked to growth, invasion, metastasis, angiogenesis of gastric carcinoma. Anticancer Res 26:3579–3584 [PubMed] [Google Scholar]
  28. Zheng H, Takahashi H, Nakajima T, Murai Y, Cui Z, Nomoto K, Tsuneyama K, Takano Y (2006b) MUC6 down-regulation correlates with gastric carcinoma progression and a poor prognosis: an immunohistochemical study with tissue microarrays. J Cancer Res Clin Oncol 132:817–823 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zheng HC, Takahashi H, Murai Y, Cui ZG, Nomoto K, Miwa S, Tsuneyama K, Takano Y (2006c) EMMPRIN expression contributes to growth and angiogenesis of gastric carcinoma: a good marker to indicate local invasion and prognosis. Br J cancer 95:1371–1378 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zheng H, Takahashi H, Murai Y, Cui Z, Nomoto K, Tsuneyama K, Takano Y (2007a) Low expression of FHIT and PTEN correlates with ma- lignancy of gastric carcinoma: tissue- array findings. Appl Immunohistochem Mol Morphol (in press) [DOI] [PubMed]
  31. Zheng H, Takahashi H, Murai Y, Cui Z, Nomoto K, Miwa S, Tsuneyama K, Takano Y (2007b) Pathobiological characteristics of intestinal- and diffuse-type gastric carcinoma in Japan: An immunostaining study on the tissue microarray. J Clin Pathol 60:273–277 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zippo A, De Robertis A, Bardelli M, Galvagni F, Oliviero S (2004) Identification of Flk-1 target genes in vasculogenesis: Pim-1 is required for endothelial and mural cell differentiation in vitro. Blood 103:4536–4544 [DOI] [PubMed] [Google Scholar]

Articles from Journal of Cancer Research and Clinical Oncology are provided here courtesy of Springer

RESOURCES