Overexpression of GRP78 and GRP94 are markers for aggressive behavior and poor prognosis in gastric carcinomas

Human Pathology (2008) 39, 1042–1049

www.elsevier.com/locate/humpath

Original contribution

Overexpression of GRP78 and GRP94 are markers for aggressive behavior and poor prognosis in gastric carcinomas☆ Hua-chuan Zheng MD, PhD a,⁎, Hiroyuki Takahashi MD, PhD b , Xiao-han Li MD b,c , Takuo Hara MD d , Shinji Masuda MD, PhD d , Yi-fu Guan PhD a , Yasuo Takano MD, PhD b a

Department of Biochemistry and Molecular Biology, College of Basic Medicine, China Medical University, Shenyang 110003, China b Department of Diagnostic Pathology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan c Division of Pathology, Shengjing Hospital of China Medical University, Shenyang 110001, China d Kouseiren Takaoka Hospital, Takaoka 933-8555, Japan Received 6 October 2007; revised 6 November 2007; accepted 13 November 2007

Keywords: Gastric carcinoma; GRP78; GRP94; Pathogenesis; Progression; Prognosis

Summary Glucose-related proteins (GRPs) are ubiquitously expressed in endoplasmic reticulum and able to assist in protein folding and assembly; consequently, they are considered as molecular chaperones. GRP78 and GRP94 expression was induced by glucose starvation and up-regulated in the malignancies. To clarify the roles of both molecules in tumorigenesis and progression of gastric carcinomas, immunohistochemistry was used on tissue microarray containing gastric carcinomas, adenomas, and nonneoplastic mucosa using the antibodies against GRP78 and GRP94, with a comparison of their expression with clinicopathological parameters of carcinomas. Gastric carcinoma cell lines (MKN28, AGS, MKN45, KATO-III, and HGC-27) were studied for both proteins by immunohistochemistry and Western blot. There was more expression of both proteins in gastric carcinoma and adenoma than in nonneoplastic mucosas (P b .05). All gastric carcinoma cell lines showed their expression at different levels. They were positively correlated with tumor size, depth of invasion, lymphatic and venous invasion, lymph node metastasis, and Union Internationale Contre le Cancer staging (P b .05), with positive relationship between both proteins (P b .05). Univariate analysis indicated the postsurgical cumulative survival rate of patients with positive GRP78 or GRP94 expression to be lower than that in those without GRP78 or GRP94 expression (P b .05), but the close link disappeared if stratified according to depth of invasion (P N .05). Multivariate analysis showed that age, depth of invasion, lymphatic invasion, lymph node metastasis, Union Internationale Contre le Cancer staging, and Lauren classification (P b .05), but not GRP78 and GRP94 expression, were independent prognostic factors for carcinomas (P N .05). Up-regulated expression of GRP78 and GRP94 was possibly involved in pathogenesis, growth, invasion, and metastasis of gastric carcinomas. They were considered objective and effective markers for the aggressive behavior and poor prognosis in gastric carcinomas. © 2008 Published by Elsevier Inc.

☆ This work was partially supported by the Japanese Ministry of Education, Science, Sports, and Culture, Grant-in-Aid for Scientific Research 14770072, 18590324 (Tokyo, Japan), the Japanese Smoking Foundation (Tokyo, Japan) and Shenyang Outstanding Talent Foundation (Shenyang, China). ⁎ Corresponding author. E-mail address: [email protected] (H. Zheng).

0046-8177/$ – see front matter © 2008 Published by Elsevier Inc. doi:10.1016/j.humpath.2007.11.009

GRP78 and GRP94 expression in gastric tumors

1. Introduction The endoplasmic reticulum (ER) plays an important role in regulating the synthesis, folding, and targeting of secretory and membrane proteins. Oxidative stress, glucose deprivation, chemical toxicity, alterations in intracellular Ca2+ levels, blockade of glycosylation, and hypoxia induce ER stress, in which expression of several ER stress-responsive genes is activated, including glucose-related proteins (GRPs) and CIEBP homologous protein (CHOP), by ER stress response element. Eukaryotic cells adapt to ER stress and thereby survive by translational attenuation to limit further accumulation of misfolded proteins, transcriptional activation of genes encoding ER-resident chaperones, and the ER-associated degradation pathway to restore the folding capacity. If the cells are exposed to prolonged or strong ER stress, the cells are destroyed by apoptosis [1-3]. GRPs are ubiquitously expressed in ER and able to assist in protein folding and assembly; consequently, they are considered as molecular chaperones. GRP78 was discovered in the late 1970s together with GRP94 as cellular proteins induced by glucose starvation and almost shows 50% homology with heat shock protein 70. In nonstressed cells, GRP78 binds to ER transmembrane sensor proteins PERK, IRE1, and ATF6 to maintain their inactive form. In addition, it is involved in protein folding in the ER lumen, translocation of newly synthesized secretory precursors across the ER membrane, presentation of aberrant proteins to the proteolytic machinery by the proteosome, and regulation of apoptosis [2,4]. GRP94 is representative of the heat shock protein 90 family of stress-induced proteins and plays an important role in stabilizing calcium homeostasis and suppressing cell death. It was found to influence the biosynthesis of such secreted and membrane-bound proteins as immunoglobulins or Tolllike receptors and also to act as a tumor vaccine, delivering the peptides for presentation to T lymphocytes [3,5]. Although GRP94 and GRP78 are localized constitutively within the ER and perform normal physiological functions under moderate levels of basal expression, they might be induced under pathological conditions, such as tumor growth or toxic damage. Tumor cells are confronted with oxygen deprivation and nutrient stress often, even with extensive angiogenesis, which causes the activation of GRPs gene expression [6,7]. The induction of GRP94 or GRP78 may be a defense mechanism for the survival of cancer cells exposed to these stress conditions. Recent studies have indicated a potential role for altered GRP78 expression and function in tumor development and progression [6-8]. Moreover, investigators have revealed that GRP78 expression is significantly high in a variety of malignancies such as breast and hepatocellular carcinoma, lung and prostate cancer, and, more recently, gastric cancer [9-12]. GRP94 overexpression was recorded in the oral carcinoma, lung cancer, and esophageal and colonic carcinomas [13-16]. Gastric carcinoma ranks as the world's second leading cause of cancer mortality behind lung cancer despite a sharp

1043 worldwide decline in both its incidence and mortality since the second half of the 20th century. Tumorigenesis and progression of gastric carcinoma are a multistage process with the involvement of a multifactorial etiology, which mainly results from gene-environment interactions [17,18]. In our study, expression of GRP78 and GRP94 was examined in gastric carcinoma, adenoma, and nonneoplastic mucosa (NNM) and compared with the clinicopathological parameters of carcinomas as well as prognosis to explore the clinicopathological significance and molecular roles of both GRPs in the stepwise development of gastric carcinomas.

2. Materials and methods 2.1. Subjects Gastric adenocarcinomas (n = 487) and NNMs (n = 86) were collected from surgical resection and gastric intestinaltype adenoma (n = 23) from endoscopic biopsy or polypectomy in our affiliated hospital and related institutes between 1993 and 2006. The patients with gastric carcinoma were 343 men and 145 women (range, 29 to 91 years, mean = 64.5 years). Among them, 185 cases have tumors accompanied with lymph node metastasis. None of the patients underwent chemotherapy or radiotherapy before surgery. 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 all patients by consulting their case documents and through telephone.

2.2. Pathology All tissues were fixed in 4% neutralized formaldehyde, embedded in paraffin, and incised into 4-μm sections. These sections were stained by hematoxylin and eosin (HE) to confirm their histologic diagnosis and other microscopic characteristics. The staging for each gastric carcinoma was evaluated according to the Union Internationale Contre le Cancer (UICC) system for the extent of tumor spread [19]. Histologic architecture of gastric carcinoma was expressed in terms of the Lauren's classification [20]. Furthermore, tumor size, depth of invasion, and lymphatic and venous invasion were determined.

2.3. Tissue microarray Representative areas of solid tumors were identified in HE-stained sections of the selected tumor cases, and a 2-mm–diameter tissue core per donor block was punched out 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

1044 polylysine-coated glass slides. HE staining was performed on tissue microarray for confirmation of tumor tissue.

2.4. Cell lines and culture Gastric carcinoma cell lines MKN28 (well-differentiated adenocarcinoma), AGS (moderately differentiated adenocarcinoma), MNK45 (poorly differentiated adenocarcinoma), KATO-III (poorly differentiated adenocarcinoma), and HGC-27 (undifferentiated adenocarcinoma) came from the Japanese Physical and Chemical Institute. They were maintained in RPMI 1640 (MKN28, MKN45, and KATOIII), minimum essential medium (HGC-27), and Ham F12 medium (AGS) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37°C. Total protein was prepared from all cells by cell disruption buffer according to PARIS manual (Arcturus Bioscience, Mountain View, CA). All cells were collected by centrifugation, rinsed with phosphate-buffered saline, fixed by 10% formalin, and then embedded in paraffin as routinely processed.

2.5. Immunohistochemistry Consecutive sections were deparaffinized with xylene, dehydrated with alcohol, and subjected to antigen retrieval by irradiating in target retrieval solution (DAKO, Carpinteria, CA) for 5 minutes in a microwave oven (Oriental Rotor, Tokyo, Japan). Five percent bovine serum albumin

H. Zheng et al. was then applied for 1 minute to prevent nonspecific binding. The sections were incubated with primary antibodies for 15 minutes and then treated with the anti-goat conjugated to horseradish peroxidase (DAKO, 1:100) antibodies for 15 minutes. All the incubations were performed in a microwave oven to allow intermittent irradiation as described previously [21]. After each treatment, the slides were washed with TBST (10 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.1% Tween 20) 3 times for 1 minute. Goat anti-GRP78 (sc-1050; Santa Cruz Biotechnology, Santa Cruz, CA; 1:50) and goat antiGRP94 (sc-1794, Santa Cruz Biotechnology, 1:50) antibodies were used to detect the individual proteins. Binding sites were visualized with 3,3′-diaminobenzidine. After counterstaining with Mayer 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 from 5 representative fields of each section blindly by 3 independent observers (Takano Y, Li XH, and Zheng HC). The positive percentage of counted cells was graded semiquantitatively according to a 4-tier scoring system: negative (−), 0% to 5%; weakly positive (+), 6% to 25%; moderately positive (++), 26% to 50%; and strongly positive (+++), 51% to 100%.

2.6. Western blot Fifty micrograms of denatured protein was separated on a sodium dodecyl sulfate–polyacrylamide gel (10% acrylamide)

Fig. 1 Immunohistochemical staining of GRP78 and GRP94 in gastric carcinoma cell lines. GRP78 was positively immunostained in the cytoplasm of MKN28 (A), AGS (C), MKN45 (E), KATO-III (G), and HGC-27(I); GRP94 in the cytoplasm of MKN28 (B), AGS (D), MKN45 (F), KATO-III (H), and HGC-27(J).

GRP78 and GRP94 expression in gastric tumors

Fig. 2 Western blot analysis of GRP78 and GRP94 in gastric carcinoma cell lines. Cell lysate (50 μg) was loaded and probed with anti-GRP78 (78 kd) and anti-GRP94 (94 kd) antibody with βactin (42 kd) as an internal control. Lane 1, MKN28; lane 2, AGS; lane 3, MKN45; lane 4, KATO-III; lane 5, HGC-27.

and transferred to Hybond membrane (Amersham, Freiberg, Germany), which was then blocked overnight in 5% skim milk in TBST. For immunoblotting, the membrane was

1045 incubated for 1 hour with the antibody against GRP78 and GRP94 as described in the part of immunohistochemistry. Afterward, it was rinsed by TBST and incubated with antigoat immunoglobulin G conjugated to horseradish peroxidase (DAKO, 1:1000) for 1 hour. All the incubations were performed in a microwave oven to allow intermittent irradiation as recommended by Li et al [22]. Bands were visualized with x-ray film (Fujifilm, Tokyo, Japan) by ECL Plus detection reagents (Amersham). After that, membrane was washed with WB Stripping Solution (pH 2-3; Nacalai, Tokyo, Japan) for 1 hour and treated as described above except that mouse anti–β-actin antibody (Sigma, 1:5000) and anti-mouse immunoglobulin G conjugated to horseradish peroxidase (DAKO, 1:1000) were used as internal controls.

Fig. 3 Immunohistochemical staining of GRP78 and GRP94 in gastric tissue samples. Note that GRP78 positivity was positively observed in the cytoplasm of gastric adenoma (B), carcinoma (C), and, occasionally, in deep propria gland (A), but not in the superficial epithelium (A). GRP94 was also expressed in the cytoplasm of gastric adenoma (E), carcinoma (F), and, occasionally, in deep propria gland (D), but not in the superficial epithelium (D).

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2.7. Statistical analysis

Table 2

Statistical evaluation was performed using the Spearman correlation test to analyze the rank data. Kaplan-Meier survival plots were generated, and comparisons between survival curves were made with the log-rank statistic. The Cox proportional hazards model was used for multivariate analysis. P less than .05 was considered as statistically significant. The SPSS 10.0 software (Chicago, IL) was used to analyze all data.

3. Results 3.1. Expression of GRP78 and GRP94 in gastric tumors As shown in Figs. 1 and 2, GRP78 and GRP94 expression was positive in the cytoplasm of MKN28, AGS, MKN45, KATO-III, and HGC-27. Both proteins were homogeneously stained in the cytoplasm of gastric propria gland, adenoma, and carcinomas (Fig. 3) and occasionally observed in the infiltrating inflammatory cells (data not shown); but there was no expression of both GRPs in gastric intestinal metaplasia, a precursor of gastric carcinomas. Overall, GRP78 expression was detected, respectively, in 23 of 86 NNMs (26.7%), 20 of 23 adenoma patients (87.0%), and 315 of 487 gastric carcinoma patients (64.7%). However, GRP78 was highly expressed in gastric adenomas and carcinomas compared with NNMs (P b .05, Table 1). GRP94 expression was positive in 298 cases of 474 gastric carcinomas (62.9%) and gastric adenoma (72.2%, 13/18), higher than in NNMs (24.1%, 20/83; P b .05; Table 2).

3.2. The relationship between the expression of GRP78 and GRP94 and clinicopathological features of gastric carcinomas As summarized in Table 3, GRP78 expression was positively linked to tumor size, depth of invasion, lymphatic and venous invasion, lymph node metastasis, and UICC staging (P b .05), but not with age or sex (P N .05). In addition, GRP94 expression was positively associated with

Table 1

GRP78 expression in gastric carcinogenesis

Groups

N

NNM Adenoma Carcinoma

86 23 487

GRP78 expression −

+

++

+++

PR (%)

63 3 172

12 2 32

8 10 67

3 8 216

26.7 ⁎ 87.0 64.7

Abbreviation: PR, positive rate. ⁎ Compared with gastric adenoma or carcinoma, P b .001.

Groups

NNM Adenoma Carcinoma

GRP94 expression in gastric carcinogenesis n

83 18 474

GRP94 expression −

+

++

+++

PR (%)

63 5 176

14 3 54

3 7 88

3 3 156

24.1 ⁎ 72.2 62.9

⁎ Compared with gastric adenoma or carcinoma, P b .001.

tumor size, depth of invasion, lymphatic and venous invasion, lymph node metastasis, and UICC staging (P b .05). GRP94 was frequently expressed in older carcinoma patients compared with the other ones (P b .05, Table 4). GRP94 preferred to be expressed in the male patients of gastric carcinoma in comparison with female ones (P b .05, Table 5). There was no difference in GRP94 or GRP78 expression between intestinal- and diffuse-type carcinomas (P N .05).

Table 3 Relationship between GRP78 expression and clinicopathological features of gastric carcinomas Clinicopathological features Age (y) b65 ≥65 Sex Male Female Tumor size (cm) b4 ≥4 Depth of invasion Tis-1 T2-4 Lymphatic invasion − + Venous invasion − + Lymph node metastasis − + UICC staging 0-I II-IV Lauren classification Intestinal type Diffuse type

n

GRP78 expression −

+

++ +++ PR (%) P .158

201 286

77 15 27 82 61.7 95 17 40 134 66.8 .085

343 113 24 46 160 67.1 144 59 8 21 56 59.0 b.001 247 105 19 31 92 57.5 240 67 13 36 124 72.1 b.001 248 109 20 32 87 56.0 239 63 12 35 129 73.6 b.001 311 130 18 43 120 58.2 176 42 14 24 96 76.1 b.001 421 158 30 60 173 62.5 66 14 2 7 43 78.8 b.001 302 127 18 39 118 57.9 185 45 14 28 98 75.7 b.001 275 121 21 35 98 56.0 212 51 11 32 118 75.9 .133 266 82 216 86

20 40 124 69.2 11 27 92 60.2

Abbreviations: Tis, carcinoma in situ; T1, lamina propria and submucosa; T2, muscularis propria and subserosa; T3, exposure to serosa; T4, invasion into serosa.

GRP78 and GRP94 expression in gastric tumors

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4. Discussion

Table 4 Relationship between GRP94 expression and clinicopathological features of gastric carcinomas Clinicopathological features Age (y) b65 ≥65 Sex Male Female Tumor size (cm) b4 ≥4 Depth of invasion Tis-1 T2-4 Lymphatic invasion − + Venous invasion − + Lymph node metastasis − + UICC staging 0-I II-IV Lauren classification Intestinal type Diffuse type GRP78 expression − +∼+++

n

GRP94 expression −

+

++ +++ PR (%) P .006

201 273

87 24 36 54 56.7 89 30 52 102 67.4 .022

329 110 37 68 114 66.6 145 66 17 20 42 54.5 b.001 239 106 36 45 52 55.6 235 70 18 43 104 70.2 b.001 240 111 38 43 48 53.8 234 65 16 45 108 72.2 b.001 304 131 38 56 170 45 16 32

79 56.9 77 73.5 b.001

408 166 49 71 122 59.3 66 10 5 17 34 84.8 b.001 294 130 42 56 180 46 12 32

66 55.8 90 25.6

269 122 40 48 205 54 14 40

59 54.6 97 73.7

255 211

84 67.8 72 58.8

b.001

.368 82 36 53 87 17 35

b.001 156 105 16 18 17 32.7 293 53 34 69 137 81.9

Both GRP78 and GRP94 play essential roles in ER linked to nuclear signaling, protein folding, sorting and secretion, protection against Ca2+ depletion stress, and antigen presentation [1-4]. Here, both molecules were found to be expressed in the cytoplasm of the gastric adenoma and carcinomas, carcinoma cell lines, consistent with their specific distribution in the ER. Statistically, GRP78 or GRP94 expression was highly expressed in gastric adenoma and adenocarcinoma in comparison with NNMs, in line with previous findings [9-16], suggesting that their up-regulated expression was involved in the pathogenesis of gastric carcinomas. Their concordant overexpression in gastric carcinomas might be due to the similar control elements of transcriptional control [23]. Glucose deprivation or hypoxia could also induce the expression of both proteins to form a protective response mechanism by which cells accommodate potentially lethal stress in the tumor microenvironment because the neovascularization could not satisfy the nutrients' demands for the growth of carcinoma cells [6,8]. GRP94 overexpression detected in rat gastric carcinomas also provided another evidence for our speculation [3]. Zheng et al [24] found that gastrointestinal adenoma and adenocarcinoma also displayed higher proliferation levels in line with elevated expression of GRP78 and GRP94 in gastric adenoma and carcinoma because of their antiapoptotic functions. Despite no statistical difference, the higher expression of both GRP78 and GRP94 in adenoma than carcinoma might be attributable to the high proliferation and low angiogenesis of adenoma and indicated that both GRPs might be early markers of adenomas and be slightly lost as the cancer progresses. Aberrant GRPs' expression and activation may play an important role in cancer development and progression

3.3. Univariate and multivariate survival analysis

Table 5 Multivariate analysis of clinicopathological variables for survival with gastric carcinomas

Follow-up information was available on 487 gastric carcinoma patients after surgical operation for periods ranging from 0.2 months to 12.2 years (median = 60.2 months) after surgical operation. Fig. 4, respectively, shows survival curves stratified according to GRP78 (A) and GRP94 (B) status for gastric carcinomas. Univariate analysis using the Kaplan-Meier method indicated cumulative survival rate of patients with weak, moderate, or strong GRP78 or GRP94 expression to be obviously lower than that of those without GRP78 or GRP94 expression (P b .05). There was no statistical difference stratified according to the depth of invasion (P N .05, data not shown). Multivariate analysis using Cox proportional hazards model indicated that age, sex, depth of invasion, lymphatic invasion, lymph node metastasis, UICC staging, and Lauren classification (P b .05), but not tumor size, venous invasion, or expression of GRP78 and GRP94, were independent prognostic factors for overall gastric carcinomas (P N .05, Table 5).

Number Clinicopathological parameters

Relative risk (95% CI)

A B C D E F G

1.865(1.290-2.698) .001 1.601(1.028-2.493) .037 1.507(0.907-2.504) .114 6.669(3.021-14.721) b.001 2.235(1.469-3.402) b.001 1.053(0.695-1.596) .808 3.155(1.684-5.909) b.001

H I J K

Age (≥65 y) Sex (female) Tumor size (≥4 cm) Depth of invasion (T2-4) Lymphatic invasion (+) Venous invasion (+) Lymph node metastasis (+) UICC staging (II-IV) Lauren classification (diffuse type) GRP78 expression (+∼+++) GRP94 expression (+∼+++)

Abbreviation: CI, confidence interval.

P

0.291(0.131-0.650) 2.104(1.412-3.135)

.003 b.001

0.910(0.573-1.446)

.690

0.937(0.597-1.473)

.780

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Fig. 4 Correlation between the status of GRP78 or GRP94 and prognosis of the gastric carcinoma patients. Kaplan-Meier curves for cumulative survival rate of patients with gastric carcinomas according to the expression of GRP78 (A) or GRP94 (B) in gastric carcinomas.

[6,9-16]. In the present study, overexpression of GRP78 and GRP94 was found to closely link to the tumor size, depth of invasion, lymphatic or venous invasion, and UICC staging, indicating that their overexpression might have impact on the growth, invasion, metastasis, and progression of gastric carcinomas and can be used to indicate the aggressive behaviors of carcinomas. High GRP94 expression in the high-staging colonic carcinoma with remote metastasis also supported our opinion about the promoting role of GRPs in the progression of gastric carcinomas [15]. Knockdown GRP78 expression inhibited gastric cell invasion in vitro and growth and metastasis, and enhanced GRP94 expression has a close relationship with cancer cell growth [10,15]. Up-regulated GRP78 expression has been implicated to enhance the ability of carcinoma cells to counterattack T-lymphocyte–mediated cytotoxicity or adverse physiological conditions [25,26]. Song et al [25] found that chronic hypoxia induced GRP78 expression in human gastric tumor cells through a protein kinase Cɛ/ERK/AP-1 signaling cascade. Some researchers reported that elevated GRP expression was significantly related to the development of

H. Zheng et al. resistance to chemotherapy and photodynamic therapy [26-34]. Therefore, it might be hypothesized that GRP overexpression forms a positive feedback as an adaptive response because a worse tumor microenvironment enhances their expression, which promotes proliferation and spreading of carcinoma cells, injures the immune attack of hosts, or makes up the molecular mechanisms of invalid treatment of malignancies. In this investigation, we analyzed the relation of GRP78 or GRP94 expression with postsurgical survival of more than 450 patients with gastric carcinoma. The results revealed a close link between their overexpression and worse survival. If stratified according to the depth of invasion, the significant correlation disappeared, indicating that the relationship between GRP78 or GRP94 expression and prognosis depends on the depth of invasion. It was previously documented that GRP78 could be used as a good marker for the worse prognosis of gastric carcinoma, hepatocelluar carcinoma, and prostate cancer [10,11,35]. It was the same for GRP94 expression in hepatocellular carcinoma, in line with our data [35]. Nevertheless, positive GRP78 expression was positively associated with the better survival of neuroblastoma patients as an independent prognostic factor [36]. The multivariate analysis demonstrated that age, sex, depth of invasion, lymphatic invasion, lymph node metastasis, UICC staging, and Lauren classification, but not expression of GRP78 and GRP94, venous invasion, and tumor size, were independent prognostic factors for carcinomas. These findings suggested that GRP78 or GRP94 expression is an indicator for the worse prognosis of gastric carcinoma patients, albeit not independent. In conclusion, up-regulated GRP78 and GRP94 expression was involved in pathogenesis of gastric carcinomas and closely related to subsequent growth, invasion, metastasis, and worse prognosis of carcinomas. They were considered objective and effective markers to indicate aggressive behavior and prognosis of gastric carcinomas.

Acknowledgment We particularly thank Kanako Yasuyoshi, Tokimasa Kumada, and Hideki Hatta for their technical help and Yukari Inoue for her secretarial assistance.

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