Ni Dai, Ai-Ping Lu, Ji-You Li, Department of Pathology, Peking University School of Oncology, Beijing Cancer Hospital, Beijing 100036, China
Cheng-Chao Shou, Department of Biochemistry & Molecular Biology, Peking University School of Oncology, Beijing Cancer Hospital, Beijing 100036, China
Author contributions: Li JY, Shou CC and Lu AP designed the research; Dai N and Lu AP performed the research; Shou CC provided the antibody; Dai N and Lu AP analyzed data; Dai N and Li JY wrote the paper.
Supported by Grant for Key Technology Research and Development Program 2002BA711A06, and National Basic Research Priorities Program 973 Project 1998051203, from the Ministry of Science and Technology of China, and grant H020920030390, from the Beijing Science and Technology Commission
Correspondence to: Ji-You Li, MD, Department of Pathology, Peking University School of Oncology, Beijing Cancer Hospital, 52 Fucheng Road, Haidian District, Beijing 100036, China. email@example.com
Telephone: +86-10-88196291 Fax: +86-10-88122450
Received: November 22, 2008 Revised: January 21, 2009
Accepted: January 28, 2009
Published online: March 28, 2009
AIM: To investigate the prognostic significance of phosphatase regenerating liver 3 (PRL-3) protein expression in gastric cancer.
METHODS: PRL-3 expression in paraffin-embedded tumor specimens from 293 patients with gastric cancer was studied retrospectively by immunohistochemistry. Monoclonal antibody specifically against PRL-3, 3B6, was obtained with hybridoma technique.
RESULTS: Positive PRL-3 expression was detected in 43.3% (127 of 293) of gastric cancer cases. High expression of PRL-3 was positively correlated with tumor size, depth of invasion, vascular/lymphatic invasion, lymph node metastasis, high TNM stage and tumor recurrence. Patients with positive PRL-3 expression had a significantly lower 5-year survival rate than those with negative expression (28.3% vs 51.9%, P < 0.0001). Patients who received curative surgery, and with positive PRL-3 expression had a significant shorter overall survival and disease-free disadvantage over patients with negative expression (hazard ratio of 16.7 and 16.6, respectively; P < 0.0001 for both). Multivariate analysis revealed that PRL-3 expression was an independent prognostic indicator for overall and disease-free survival of gastric cancer patients, particularly for survival in TNM stage Ⅲ patients.
CONCLUSION: PRL-3 expression is a new independent prognostic indicator to predict the potential of recurrence and survival in patients with gastric cancer at the time of tumor resection.
© 2009 The WJG Press and Baishideng. All rights reserved.
Key words: Phosphatase regenerating liver 3; Gastric cancer; Prognosis; Recurrence; Antibody
Peer reviewers: Harry HX Xia, PhD, MD, Novartis Pharmaceuticals Corporation, One Health Plaza, East Hanover, NJ 07936-1080, United States; Toru Ishikawa, MD, Department of Gastroenterology, Saiseikai Niigata Second Hospital, Teraji 280-7, Niigata, Niigata 950-1104, Japan
Dai N, Lu AP, Shou CC, Li JY. Expression of phosphatase regenerating liver 3 is an independent prognostic indicator for gastric cancer. World J Gastroenterol 2009; 15(12): 1499-1505 Available from: URL: http://www.wjgnet.com/1007-9327/15/1499.asp DOI: http://dx.doi.org/10.3748/wjg.15.1499
Gastric cancer (GC) is one of the most common malignancies in the world with a high incidence and death rate. In China, it remains the most frequent cancer and the second cancer-related cause of death with a high case fatality. TNM staging system is used worldwide to predict prognosis and direct therapeutic decisions of patients with GC. The 5-year survival rate in patients with stage Ⅰ GC is close to 90% and around 10% for patients with stage Ⅳ GC. However, the prognoses of patients with stage Ⅱ and Ⅲ GC are more heterogeneous and less predictable by staging criteria. Therefore, finding molecular markers that are able to predict the potential of tumor recurrence and prognosis of patients is extremely important for appropriate individualized therapy. Phosphatase regenerating liver 3 (PRL-3) (also known as PTP4A3) belongs to a newly discovered group of phosphotase regenerating liver family which is implicated in oncogenic and metastatic processes[3,4]. The PRL family represents a protein tyrosine phosphatase superfamily possessing a unique COOH-terminal prenylation motif and a protein tyrosine phosphatase-active site signature sequence CX5R[5,6]. Three PRLs (PRL-1, -2, and -3) are highly homologous with similar amino acid sequence of 76%-87%[7-9]. Recent reports found that PRL-3 was consistently expressed at higher level of metastasis in liver compared to normal colorectal epithelia and primary cancer tissues[10,11]. A growing body of evidence showed that an excess of PRL-3 phosphatase is a key alteration contributing to the acquisition of metastatic properties of the tumor cells. For example, nontumorigenic or low metastatic cell lines transfected with wild type PRL-3 displayed higher cell motility and invasiveness and could induce metastatic tumor formation in mice, while cells expressing catalytically inactive mutant PRL-3 significantly reduced the migratory capability[12,13]. Knockdown of endogenous PRL-3 in cancerous cells using small interfering RNA or phosphatase inhibitors can abrogate cell motility and the ability to form metastasis-like tumors in mice[12-15]. PRL-3 was further demonstrated to be a useful indicator for tumor recurrence and patient outcome in several human cancers including colorectal cancer and breast cancer[16-19]. In gastric cancer, PRL-3 was found to be highly expressed in tumor metastatic lymph nodes and closely associated with the peritoneal metastasis[20-23], but the prognostic impact of PRL-3 expression in gastric cancer still remains to be further investigated.
In this study, we detected the expression of PRL-3 in GC tissue samples by immunohistochemistry using a PRL-3 specific monoclonal antibody 3B6 to investigate PRL-3 protein expression in GC tissues and whether PRL-3 could be applied as a prognostic indicator for GC to predict the potential of tumor recurrence and patient outcome.
MATERIALS AND METHODS
This retrospective study enrolled patients who underwent clinical surgery for primary gastric cancer at the Department of Surgery, Beijing Cancer Hospital, Peking University School of Oncology between July 1994 and December 2000. Patients with inadequate histologic specimens or missing clinical information were excluded. A total of 293 patients were finally included. There were 194 males and 99 females, with ages ranging from 25 to 82 (mean ± SD, 58 ± 17.1 years). Two hundred (68.3%) patients received curative resection (R0) with radical lymph node dissection; the remaining 93 (31.7%) patients with microscopic or macroscopic tumor residues were given palliative resection (R1/R2). Site distribution of the primary tumor was 153 at antrum, 52 at cardia or fundus, and 88 at corpus. Tumor size ranged from 5 to 120 mm (mean, 43.8 mm).
Data were collected from clinical case report record and follow-up database. Tumor staging was based on the clinical evaluation and postoperative pathological reports. TNM staging was on the basis of the 1997 fifth edition of AJCC/UICC TNM staging criteria for gastric cancer. The tumors were histologically classified according to the WHO classification criteria.
None of these patients had received radiotherapy or chemotherapy preoperatively. All the patients were followed up at regular intervals of 6 mo after surgery until June 2006 with a minimum of five years. Tumor recurrence was clinically defined as the reappearance of tumor after curative surgery. The overall survival time was calculated from the date of surgery to the date of last visit or death and the disease-free survival time from the date of resection to relapse.
Immunostaining of PRL-3
Tumor tissue specimens from the 293 patients were routinely fixed in 10% formalin and embedded with paraffin. Paraffin embedded tissue samples were cut into 4 m sections. The sections were put in an oven at 60℃ for 5 h and cooled down overnight before they were deparaffinized in xylene. The sections were then dehydrated in a graded ethanol series, and treated with 3% hydrogen peroxide solution for 10 min to block endogenous peroxidase activity. Antigen retrieval was performed by microwaving the sections in 1 mmol/L EDTA (PH 8.0) for 15 min. PRL-3 monoclonal antibody 3B6 (a generous gift from Prof. Shou, Beijing Institute for Cancer Research, China) was used as the primary antibody at a dilution of 1:100 overnight at 4℃. The Powervision two-step histostaining reagent PV-6002 (Dako, Glostrop, Denmark) was applied as the secondary antibody. The sections were visualized with diaminobenzidine and counterstained with hematoxylin. Each incubation step was followed by washing with phosphate-buffered saline. For negative control, the primary antibody was omitted from the reaction sequence. Sections of liver metastasis from colon cancer with known strong PRL-3 immunoreactivity were used as positive controls. The number of tumor cells with cytoplasm staining of PRL-3 was counted without knowledge of the clinicopathological data, and > 5% positive tumor cells were defined as positive PRL-3 expression.
Statistical analyses were performed with SAS 8.1 software. The c2 test was used to analyze the association between PRL-3 expression and clinicopathological features of GC. Cumulative survival rates and differences in survival curves were estimated by Kaplan-Meier method with the log-rank test. The effect of PRL-3 on survival was analyzed using the Cox proportional hazard regression model adjusted for clinical and histopathologic features. Two-sided P values of less than 0.05 were considered to be statistically significant.
Forty-two patients were classified as stage Ⅰ, 52 as stage Ⅱ, 99 as stage Ⅲ and 100 as stage Ⅳ. A total of 194 cases were poorly differentiated, 69 cases were moderately differentiated and the remaining 30 cases were well differentiated.
The follow-up period for survivors ranged from 2 to 120 mo (median, 31 mo). The 5-year overall survival rate was 41.7% in the entire cohort of patients, 92.9% in stage Ⅰ, 72.5% in stage Ⅱ, 32.5% in stage Ⅲ and 12.3% in stage Ⅳ patients. One hundred and five patients remained alive and disease-free, 15 patients were alive with disease. One hundred and seventy patients died of GC, and 3 patients died of other causes. Among the 200 patients who received curative surgery, 23 patients had tumor recurrence with 3 in peritoneum, 3 in lymph node, 7 in liver, 3 in other organs (2 ovarian, 1 lung), 4 in multiple organs and 3 in remnant stomach.
PRL-3 expression in GC and its relation with clinicopathological features
PRL-3 immunostaining was predominantly localized in the cytoplasm of normal or tumor epithelial cells. PRL-3 stained cells in normal epithelia were mainly observed in the neck of gastric glands (Figure 1). Among the 293 GC specimens analyzed, 127 (43.3%) tumors had positive PRL-3 expression. The rate of positive PRL-3 expression was significantly higher in stage Ⅲ and Ⅳ than in stage Ⅰ and Ⅱ (48.7% vs 31.9%, P = 0.007). High expression of PRL-3 was correlated closely with large tumor size, depth of invasion in gastric wall, lymph node metastasis, vascular/lymphatic invasion and recurrent frequency. No significant correlation was observed between PRL-3 expression and sex, age, distant metastasis, grade of differentiation and surgical curability (Table 1).
Univariate survival analysis of prognostic impact of PRL-3 expression
Kaplan-Meier method with log-rank test revealed that patients with positive PRL-3 expression had a significantly lower cumulative 5-year overall survival rate than those with negative expression (28.3% vs 51.9%, P < 0.0001). Among the 99 patients with stage Ⅲ GC, those with positive PRL-3 expression had a lower survival rate than those with negative expression (18.6% vs 43.2%, P = 0.0004, Figure 2). Among the 200 patients who received curative surgery, patients whose tumor had positive PRL-3 expression had worse disease-free status and poorer overall survival (hazard ratio, 16.6 and 16.7 respectively; P < 0.0001 for both) than those with negative expression (Figure 3). Among patients who received palliative resection or patients in stages other than stage Ⅲ, PRL-3 showed no significant correlation with prognosis.
Multivariate survival analysis of prognostic impact of PRL-3 expression
Multivariate analysis by extended Cox regression model revealed that PRL-3 expression remained an independent prognostic factor after adjusting for sex, age, tumor location, tumor size, depth of invasion, lymph node metastasis, distant metastasis, TNM staging, vascular/lymphatic invasion, and surgical curability. PRL-3 expression was a significantly independent prognostic factor for the overall survival of all 293 GC patients. For the 200 patients who received curative resection, PRL-3 expression was found to be an independent prognostic factor for both disease-free and overall survival. The results are shown in Table 2.
In this study, we detected the protein expression of PRL-3 in GC tissues using the highly specific monoclonal antibody 3B6 prepared by Peng et al. PRL-3 had higher rates of positive expression in advanced stages and PRL-3 expression was positively correlated with tumor size, depth of invasion, and lymph node metastasis vascular/lymphatic invasion at the time of surgery and recurrence. These results suggest that PRL-3 may play a crucial role in invasion, progression and metastasis of GC. The present analyses revealed that PRL-3 was an independent prognostic indicator for overall and disease-free survival of GC. Among patients with advanced TNM stages especially stage Ⅲ, patients with positive PRL-3 expression have more frequent recurrence and poorer survival, adjuvant therapies such as radiotherapy and chemotherapy may be necessary after curative surgery. Evaluation of PRL-3 expression status may identify a subset of patients with GC who require more intensive treatment.
Miskad et al reported that PRL-3 was highly expressed in metastatic lymph nodes of GC and high expression of PRL-3 was closely associated with tumor stage. Wang et al found that the high expression of PRL-3 in lymph node metastases had a negative impact on the prognosis of patients with GC. Li et al[22-23] reported that PRL-3 expression was correlated with peritoneal metastasis and poor prognosis in GC patients. The superiority of our study may be the use of antibody specifically against PRL-3 and the relatively extensive clinical data which facilitated the analysis from multiple angels. One limitation of our study is that the relatively small sample hindered the analysis in stage Ⅰ and stage Ⅱ patients.
Attributed to the high sequence similarity of three PRLs and the wide expression of PRL-1 and PRL-2 in normal tissues and cancer cell lines, commercial polyclonal antibody against PRL-3 used in previous studies could potentially cross-react with PRL-1 and PRL-2. Monoclonal antibody specifically reacting with PRL-3 is extremely important to exclude the interference of PRL-1 and PRL-2 and therefore allows us to accurately evaluate the prognostic implication of PRL-3 expression[25,26]. To prepare specific PRL-3 monoclonal antibody, Peng et al obtained the monoclonal antibody 3B6 with hybridoma technique, and confirmed its specificity with ELISA and Western blotting assays. High specificity of the monoclonal antibody 3B6 against PRL-3 was demonstrated. The applicability of the monoclonal antibody has been further confirmed by two other studies investigating the prognostic impact of PRL-3 expression in colorectal cancer and breast cancer[16,19].
PRL-3 has been confirmed to be an important metastatic instrumental molecule. Although the actual signal transduction pathways in which PRL-3 is implicated are largely unknown, Rho signaling pathway molecules which are regulators of motility and invasion have been identified as potential candidate targets of PRL-3. PRL-3 transfectants displayed altered extracellular matrix adhesive property and up-regulated integrin-mediated cell spreading efficiency[14,27]. Peng et al recently found that PRL-3 activates the mitogen-associated protein kinase pathway by binding a cell membrane protein in cell migration and invasion. PRL-3 was also found to be associated with membrane structures including ruffles, protrusions, and some vacuolar-like membrane extensions which have been demonstrated to play a role in cell movement and invasion[29-31]. Besides, PRL-3 may be involved in triggering angiogenesis and establishing microvasculature in vitro[32-34]. These findings suggest that PRL-3 may be implicated with the key steps of tumor metastasis including tumor cell invasion and survival in circulation and vasculature formation.
In addition to its role in predicting tumor recurrence and prognosis, PRL-3 has a potential value of being a candidate for metastasis tailored therapies. Since primary tumors can be surgically resected, the metastatic tumors are the main cause responsible for a high case fatality. PRL-3 was highly expressed in tumor metastasis and found to play a key role in tumor metastatic process[10-21]. PRL-3 may serve as a potential therapeutic target for cancer metastases. Inhibition of PRL activity might be carried out using phosphatase inhibitors targeting the consensus phosphatase motif, farnesyltransferase inhibitors, interference RNA or monoclonal antibody as well[9,35-37]. Recent progress in active recombinant PRL-3 production and findings on PRL-3 structure will undoubtedly facilitate the development of PRL-3 inhibitors[38-40]. Detection of PRL-3 expression would be able to provide supportive information for anti-cancer therapy.
In conclusion, PRL-3 is closely associated with tumor invasion and lymphatic metastasis and is identified as a new prognostic indicator to predict tumor recurrence and patient survival in GC.
It is established that phosphatase regenerating liver 3 (PRL-3) is consistently expressed in liver metastasis of colon cancer. A recent study reported that PRL-3 expression was related to peritoneal metastasis of stomach cancer.
In a few studies, the association of PRL-3 expression with prognosis of cancers was investigated and demonstrated that this is related to poor prognosis of breast and stomach cancer. The antibody used in many studies was commercial polyclonal antibody that could cross-react with PRL-1 and PRL-2.
Innovations and breakthroughs
A recent study showed that PRL-3 was expressed in 70.4% of 637 GCs. PRL-3 expression was correlated with peritoneal metastasis. Patients with PRL-3 negative expression had a better survival rate than those with positive PRL-3 at all stages. In this study, PRL-3 was found to express in 43.3% (127/293) of primary tumor tissues of GC. PRL-3 protein expression was demonstrated to be an independent predictor for poor prognosis in GC. In addition, a specific monoclonal antibody to PRL-3 was used, allowing us to accurately evaluate the prognostic significance of PRL-3 expression in GC.
Defection of PRL-3 protein expression in primary tumor tissues of GC might be helpful in identification of GC patients with poor prognosis who should receive more intensive treatment. In addition, PRL-3 may serve as a potential therapeutic target for GC.
PRL-3 (also known as PTP4A3) is a member of phosphate of regeneration liver (PRL) family including PRL-1, PRL-2 and PRL-3 which are implicated with oncogenic and metastatic processes of tumors. The excess of PRL-3 phosphates is an alteration contributing to the acquisition of metastatic properties of tumor cells. The monoclonal antibody 3B6 used in this study is a specific monoclonal antibody to PRL-3 generated by Peng at al, Beijing Institute for Cancer Research.
The authors examined the expression of PRL-3 protein in primary tumor tissues of 293 cases of GC. It revealed that patients with negative PRL-3 expression had a higher 5-year survival rate than those with positive PRL-3 expression. PRL-3 is an independent prognostic indicator for GC. The findings are of clinical significance.
1 Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005; 55: 74-108 PubMed
Maehara Y, Oshiro T, Endo K, Baba H, Oda S, Ichiyoshi Y, Kohnoe
S, Sugimachi K. Clinical significance of occult
Diamond RH, Cressman DE, Laz TM, Abrams CS, Taub R. PRL-1, a
unique nuclear protein tyrosine phosphatase, affects
Cates CA, Michael RL, Stayrook KR, Harvey KA, Burke YD, Randall
SK, Crowell PL, Crowell DN. Prenylation of oncogenic
Matter WF, Estridge T, Zhang C, Belagaje R, Stancato L, Dixon J,
Johnson B, Bloem L, Pickard T, Donaghue M, Acton S,
Zeng Q, Si X, Horstmann H, Xu Y, Hong W, Pallen CJ. Prenylation-dependent
association of protein-tyrosine
Saha S, Bardelli A, Buckhaults P, Velculescu VE, Rago C, St Croix
B, Romans KE, Choti MA, Lengauer C, Kinzler KW,
Bardelli A, Saha S, Sager JA, Romans KE, Xin B, Markowitz SD,
Lengauer C, Velculescu VE, Kinzler KW, Vogelstein B.
Zeng Q, Dong JM, Guo K, Li J, Tan HX, Koh V, Pallen CJ, Manser E,
Hong W. PRL-3 and PRL-1 promote cell migration,
Guo K, Li J, Tang JP, Koh V, Gan BQ, Zeng Q. Catalytic domain of
PRL-3 plays an essential role in tumor metastasis:
Molleví DG, Aytes A, Padullés L, Martínez-Iniesta M, Baixeras N,
Salazar R, Ramos E, Figueras J, Capella G, Villanueva
Peng L, Ning J, Meng L, Shou C. The association of the expression
level of protein tyrosine phosphatase PRL-3 protein
Kato H, Semba S, Miskad UA, Seo Y, Kasuga M, Yokozaki H. High
expression of PRL-3 promotes cancer cell motility and
Radke I, Götte M, Kersting C, Mattsson B, Kiesel L, Wülfing P.
Expression and prognostic impact of the protein tyrosine
Wang L, Peng L, Dong B, Kong L, Meng L, Yan L, Xie Y, Shou C.
Overexpression of phosphatase of regenerating liver-3
Wang Z, He YL, Cai SR, Zhan WH, Li ZR, Zhu BH, Chen CQ, Ma JP,
Chen ZX, Li W, Zhang LJ. Expression and prognostic
Li Z, Zhan W, Wang Z, Zhu B, He Y, Peng J, Cai S, Ma J.
Inhibition of PRL-3 gene expression in gastric cancer cell line
Li ZR, Wang Z, Zhu BH, He YL, Peng JS, Cai SR, Ma JP, Zhan WH.
Association of tyrosine PRL-3 phosphatase protein
24 Sobin LH, Fleming ID. TNM Classification of Malignant
Tumors, fifth edition (1997). Union Internationale Contre le
26 Li J, Guo K, Koh VW, Tang JP, Gan BQ, Shi H, Li HX, Zeng Q.
Generation of PRL-3- and PRL-1-specific monoclonal
28 Peng L, Jin G, Wang L, Guo J, Meng L, Shou C.
Identification of integrin alpha1 as an interacting protein of protein
31 Small JV, Stradal T, Vignal E, Rottner K. The lamellipodium: where motility begins. Trends Cell Biol 2002; 12: 112-120
33 Parker BS, Argani P, Cook BP, Liangfeng H, Chartrand SD,
Zhang M, Saha S, Bardelli A, Jiang Y, St Martin TB, Nacht M,
34 St Croix B, Rago C, Velculescu V, Traverso G, Romans KE,
Montgomery E, Lal A, Riggins GJ, Lengauer C, Vogelstein B,
35 Pathak MK, Dhawan D, Lindner DJ, Borden EC, Farver C, Yi T.
Pentamidine is an inhibitor of PRL phosphatases with
37 Guo K, Tang JP, Tan CP, Wang H, Zeng Q. Monoclonal
antibodies target intracellular PRL phosphatases to inhibit cancer
38 Sager JA, Benvenuti S, Bardelli A. PRL-3: a phosphatase for metastasis? Cancer Biol Ther 2004; 3: 952-953
39 Kim KA, Song JS, Jee J, Sheen MR, Lee C, Lee TG, Ro S, Cho
JM, Lee W, Yamazaki T, Jeon YH, Cheong C. Structure of
40 Kozlov G, Cheng J, Ziomek E, Banville D, Gehring K, Ekiel
I. Structural insights into molecular function of the metastasis-
S- Editor Tian L L- Editor Ma JY E- Editor Yin DH