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Overexpression of phosphatase regenerating liver 3 in oesophageal squamous cell carcinoma associated with metastasis and its comparison with phosphatase regenerating liver 1
Xi Lou*1, Yuqiong Liu†1, Guo Ke*, Jun‑Yi Lei‡2 and Huixiang Li†2
*Institute of Molecular and Cell Biology, Proteos, Singapore, †Department of Pathology, The First Affiliated Hospital, Zhengzhou University School of Medicine, Zhengzhou 450052, People's Republic of China, and ‡Jdxpath, Collegeville, PA, U.S.A.
Expression of PRL3 (phosphatase of regenerating liver 3) protein was examined with immunohistochemistry in 60 cases of ESCC (oesophageal squamous cell carcinoma) with matched lymph node metastasis (n = 40) and 6 cases of oesophageal adenocarcinoma. Its associations with PRL1 and clinicopathological parameters were analysed. The results showed the frequency of PRL3 protein expression was significantly higher in ESCC (39/60, 65%) than in normal oesophageal mucosa (0/20, P<0.001); higher in ESCC with lymph node metastasis (30/40, 75%) than in ESCC without lymph node metastasis (9/20, P = 0.022), as well as higher in metastatic ESCC in lymph node (38/40, 95%) than in the primary ESCC (39/60, 65%, P<0.001). PRL3 was expressed in 1 out of 6 oesophageal adenocarcinomas, but showed no nuclear staining of PRL1. Expression of PRL3 protein was positively associated with the grade and partially with the stage of ESCC. These results suggest that expression of PRL3 protein may be involved in the metastasis of ESCC and serve as a biomarker for prediction of ESCC metastasis.
Key words: oesophagus, immunohistochemistry, metastasis, PRL3, squamous cell carcinoma
Abbreviations: ESCC, oesophageal squamous cell carcinoma, PRL3, phosphatase of regenerating liver 3
1These two authors contributed equally to this work.
2Correspondence may be addressed to either of these authors (email email@example.com or firstname.lastname@example.org).
PRL (phosphatase of regenerating liver) is a new subclass (Cates et al., 1996; Zeng et al., 1998) of protein tyrosine phosphatases superfamily which is involved in numerous essential events in eukaryotes (Hunter, 2000). PRL has 3 members so far, PRL1, PRL2 and PRL3 (Cates et al., 1996; Zeng et al., 1998) and they are expressed in many normal tissues at different levels (Diamond et al., 1994). PRL3 is overexpressed in human colorectal, gastric, hepatic, ovarian, cholangiocarcinoma, nasopharyngeal and breast cancers (Ren et al., 2009; Zhou et al., 2009; Xu et al., 2010) and also closely associate with the metastasis of human cancers including colorectal, gastric, breast and oesophageal cancers (Saha et al., 2001; Kato et al., 2004; Radke et al., 2006; Ooki et al., 2010). Experiments have also shown that PRL can promote cell proliferation, enhance cell mobility, and induce metastatic tumour formation (Diamond et al., 1994; Zeng et al., 2003).
Oesophageal carcinoma is one of the most common human malignant tumour worldwide (Ribeiro et al., 1996). The incidence of ESCC (oesophageal squamous cell carcinoma) in China is up to 50/105 with mortality above 100/105 (Li and Lu, 1996; Gabbert et al., 2000). Early diagnosis is a prerequisite for effective treatment, which gives >90% of the patients survival of 5–10 years (Li and Lu, 1996). Deep invasion and metastasis are the main causes of death for patients with oesophageal carcinoma. To improve the prognosis of patients, it is important to understand, prevent and effectively treat metastasis of ESCC. We have examined PRL3 expression in ESCC and compared with expression of PRL1.
2. Materials and methods
2.1. Tissue samples
All tissue samples of ESCC were selected from surgical pathology files in the Department of Pathology, Anyang Tumor Hospital, Henan, China. Out of 60 cases in total, 40 cases had matching metastatic lymph nodes and 20 had normal oesophageal mucosa taken from resection margins. The diagnoses of ESCC were reconfirmed histologically for the tumours. The classification and clinical stage of ESCC were based on World Health Organization criteria (Gabbert et al., 2000). Among 60 ESCCs, 11, 34 and 15 are well, moderately, and poorly differentiated respectively. Three cases were stage I, 23 were stage II and 34 were stage III.
Twelve cases had tumour in the upper, 39 in the middle and 8 in the lower 1/3 of the oesophagus. Six cases of oesophageal adenocarcinoma were also chosen, classified and staged in the same way. Among 6 cases of oesophageal adenocarcinomas, 1 and 5 are well and poorly differentiated respectively.
The patients consisted of 38 men and 22 women, with ages ranging from 43 to 77 years old (mean 61). None of patient received therapy prior surgery. Informed consent was obtained from all of patients. The study was approved by the institutional review board.
Immunohistochemistry was carried out as previously described (Liu et al., 2011). The monoclonal antibody against PRL3 was used at 1:50 dilution. Five hundred cells from 5 representative areas were counted in each section. An average of 10% or more tumour cells staining was defined as positive. Immunohistochemical staining was interpreted independently by two observers (L.H.X. and L.Y.Q.), with a reproducibility of 94% (62/66). The cases with disagreement were discussed by the 2 observers and categorized according to consensus.
2.3. Statistical analyses
All data were analysed with SPSS version 11.0 statistical package (Shengce Software Ltd). The differences in positive rates and means were analysed by χ2 and t test respectively. The relationship of 2 variables was analysed by correlation analysis. P<0.05 was taken as statistically significant.
3.1. Expression of PRL3 proteins in benign oesophageal tissue
In normal oesophageal epithelium, PRL3 protein was expressed in the stratum spinosum and above, but not in the basal cells (Figure 1). Therefore, the staining of basal cell layers was used as the index of PRL3 expression in benign oesophageal tissue.
3.2. Expression of PRL3 protein in oesophageal neoplasm
In ESCC, PRL3 was expressed in 65% (39/60) of ESCC (Figure 2), significantly higher than that in normal oesophageal mucosa (0%; P<0.001, Table 1). To explore the association of PRL3 protein with tumour metastasis, we compared the frequency of PRL3 protein expression in primary ESCC with and without lymph node metastasis. PRL3 protein was expressed in 75% (30/40) of ESCC with lymph node metastasis (Figure 3), significantly higher than that of ESCC without lymph node metastasis (45%, 9/20; P = 0.022, Table 1).
Table 1 Frequency of PRL3 protein expression in benign and malignant oesophageal tissues
ESCC, oesophageal squamous cell carcinoma; LN, without lymph node metastatsis; LN+, with lymph node metastatsis; MESCC, metastatic ESCC; EA, oesophageal adenocarcinoma; *, Compared with ESCC; @, compared with ESCC (LN-); †, compared with ESCC (LN+).
Furthermore, PRL3 protein was expressed in 95% (38/40) of the metastatic ESCC in lymph nodes, significantly higher than in the primary ESCC in 3 categories [75% (30/40) in the primary ESCC with lymph node metastasis (P = 0.01); 45% (9/20) in the primary ESCC without lymph node metastasis (P<0.01); and 65% (39/60) in all of 60 primary ESCC (P<0.01, Table 1)]. The percentage and density of PRL3 stain showed no significant differences between any groups of ESCC. Out of 6 cases of oesophageal adenocarcinoma, PRL3 showed cytoplasmic staining in 1 (16.7%) well differentiated (Figure 4), but negative in 5 poorly differentiated oesophageal adenocarcinoma, significantly lower than in ESCC (P = 0.03, Table 1). No nuclear staining of PRL3 antibody was identified in any of 6 oesophageal adenocarcinomas.
3.3. Association of PRL3 expression with clinicopathological parameters in ESCC
PRL3 protein was expressed in 33.3% of poorly differentiated ESCC, significantly lower than that of well (72.7%, P = 0.047) and moderately (76.5%, P = 0.04, Table 2) differentiated ESCC. PRL3 protein was expressed in 76.5% (26/34) of ESCC in stage III, significantly higher than that of ESCC in stage IIa [47.1 (8/17), P = 0.04, Table 2], but not ESCC in stage I and IIb. PRL3 protein expression in ESCC was not associated with the patients' sex, age or tumour location (Table 2).
Table 2 Association of PRL3 protein expression frequency with clinicopathological parameters
*, compared with the second group; †, compared with the third group.
3.4. Correlation of protein expression between PRL3 and PRL1 in ESCC
To determine the correlation of expression of PRL3 protein with that of PRL1, the data were compared with PRL1's previously reported (Liu et al., 2011). The comparison in ESCC with lymph node metastasis showed no significant difference in frequencies, but a positive correlation of PRL3 protein expression with that of PRL1 (Table 3). The same results were also found in ESCC without lymph node metastasis, primary ESCC and metastatic ESCC (data not shown).
Table 3 Correlation of expression of PRL3 protein with that of PRL1 in ESCC
rs: Pearson's R-spearman correlation. P, significant positive correlation; P*, significant difference in frequency of PRL3 and PRL1 protein expression, calculated by McNemar test.
After its discovery, PRL promptly drew interest in its close association with human cancer and tumour metastasis (Bardelli et al., 2003; Radke et al., 2006; Miskad et al., 2007). We found PRL3 protein was expressed in 65% (39/60) of primary ESCC, similar to the value (78%) reported by Ooki et al. (2010). Further comparison showed that the frequency of PRL3 protein expression was significantly higher in ESCC with lymph node metastasis than in ESCC without lymph node metastasis, and also significantly higher in metastatic ESCC in lymph node than in primary ESCC. Our results are consistent with those reported for other human cancers and suggest that PRL3 may be involved in promoting the metastasis of ESCC and might serve as a good biomarker to predict the aggressiveness, especially the potential of metastasis, of ESCC.
Expression of PRL3 is positively correlated with the stage of colorectal, gastric, and ovarian cancers (Buffart et al., 2005; Polato et al., 2005; Miskad et al., 2007), and inversely correlated with the disease-free survival in breast cancer (Radke et al., 2006). In addition, PRL3 expression was also closely associated with tumour size, lymphatic and venous invasion, and extent of lymph node metastasis in gastric cancer (Miskad et al., 2007). PRL3 was also the strong independent predictor for lymph node metastasis of ESCC (Ooki et al., 2010). Our results also show a significant association of PRL3 protein expression with grade of ESCC and the stage of ESCC (between stage III and IIa). The lack of association between stage III and stage I or IIb may be due to the limited size of the samples. Thus PRL3 may serve as the predictor of prognosis of the patients with ESCC.
Despite the high level of identity in their amino acid sequences (75–86%) (Zhou et al, 2003), human PRLs are preferentially expressed in the different normal tissues (Diamond et al., 1994; Zeng et al., 1998). Such preferential expression of PRL has also been demonstrated in tumours. Radke et al. (2006) in a simultaneous study on multiple PRLs found that only PRL3 mRNA, not PRL1 or PRL2, was overexpressed in breast cancer. However, we found that mRNA expression of both PRL1 and PRL3 were significantly associated metastasis of ESCC (Liu et al., 2008). To fill the blank of a parallel study on PRL1 and PRL3, expression of both molecules in ESCC was examined and in exactly the same tissue samples and materials. The results showed no significant difference in, but excellent correlation of, expression of both proteins in every group of ESCC, including ESCC with lymph node metastasis, ESCC without lymph node metastasis, primary ESCC and metastatic ESCC in lymph node. Their expressions were significantly associated with the metastasis, the stage and the grade (PRL3 only) of ESCC. However, in oesophageal adenocarcinoma PRL3 protein was significantly less expressed than PRL1 and did not show nuclear staining of PRL1. These results suggest that both PRL1 and PRL3 serve as markers to predict the metastasis of ESCC, but only PRL1 has the potential to be used in the differential diagnosis of poorly differentiated ESCC from poorly differentiated oesophageal adenocarcinoma.
The potential of PRL3 as the therapeutic target for cancer has been proved experimentally. The cell migratory ability could be significantly reduced with catalytically inactive PRL3 mutant and even reversed by specific anti-sense oligodeoxynucleotide and phosphatase inhibitors (Wu et al., 2004; Ruan et al., 2011). PRL3-specific knockdown using siRNA (small interfering RNA) severely impaired the growth of ovarian cancer cells (Polato et al., 2005), abrogated motility (in vitro) and hepatic colonization (in vivo) of human colon cancer DLD-1 cells (Kato et al., 2004), and repressed anchorage-independent colony formation, and invasion and augmented 5-FU-induced apoptosis in all the tested ESCC cell lines with PRL3 overexpression (Ooki et al., 2010). The anti-protozoa drug, Pentamidine, an inhibitor of PRLs, can inactivate exogenous PRLs with a long effective duration (>24 h) after a pulse cell treatment at its therapeutic dose. The growth of WM9 human melanoma tumours could also be markedly inhibited by Pentamidine at a tolerable dose in nude mice with the induction of tumour cell necrosis (Pathak et al., 2002). The extremely high frequency of PRL1 and PRL3 protein expression in metastatic ESCC (95–97.5%) in lymph nodes identified in this study will provide a solid base for exploring such possibility to prevent and treat metastasis of ESCC in future.
Xi Lou performed the immunohistochemical stains, analysed data, wrote the results including Tables and Figures and proofread the paper. Yuqiong Liu collected material and clinicopathological information, assisted in data analysis, statistical analysis, and composition of Tables and proofread the paper. Ke Guo provided a monoclonal antibody against PRL 1 and 3, troubleshooted the problems of IHC stains and proofread the paper. Junyi Lei consulting on data analysis and paper writing, and proofread the paper. Huixiang Li designed the experiment, guided the experiments, guided the data analysis, and wrote the paper.
This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
Cates, CA, Michael, RL, Stayrook, KR, Harvey, KA, Burke, YD and Randall, SK (1996) Prenylation of oncogenic human PTP(CAAX) protein tyrosine phosphatases. Cancer Lett 110, 49-55
Gabbert, HE, Shimoda, T, Hainaut, P, Nakamura, Y, Field, JK and Inoue, H (2000) Squamous cell carcinoma of the esophagus. In WHO classification of tumors: tumors of the digestive system (Hamilton SR and Aaltonen, LA, eds), pp. 11-19, IARC Press, Lyon, France
Kato, H, Semba, S, Miskad, UA, Seo, Y, Kasuga, M and Yokozaki, H (2004) High expression of PRL3 promotes cancer cell motility and liver metastasis in human colorectal cancer: a predictive molecular marker of metachronous liver and lung metastases. Clin Cancer Res 10, 7318-28
Liu, YQ, Lou, X, Guo, K, Lei, JY and Li, H (2011) Expression of phosphatase regenerating liver-1 in esophageal squamous cell carcinoma associated with metastasis. ISRN Pathol. 2011;2011:Article ID 680890, 6 pages, doi:10.5402/2011/680890
Miskad, UA, Semba, S, Kato, H, Matsukawa, Y, Kodama, Y and Mizuuchi, E (2007) High PRL3 expression in human gastric cancer is a marker of metastasis and grades of malignancies: an in situ hybridization study. Virchows Arch 450, 303-10
Ooki, A, Yamashita, K, Kikuchi, S, Sakuramoto, S, Katada, N and Watanabe, M (2010) Phosphatase of regenerating liver 3 as a convergent therapeutic target for lymph node metastasis in esophageal squamous cell carcinoma. Int J Cancer 127, 543-54
Ren, T, Jiang, B, Xing, X, Dong, B, Peng, L and Meng, L (2009) Prognostic significance of phosphatase of regenerating liver-3 expression in ovarian cancer. Pathol Oncol Res 15, 555-60
Radke, I, Gotte, M, Kersting, C, Mattsson, B, Kiesel, L and Wülfing, P (2006) Expression and prognostic impact of the protein tyrosine phosphatases PRL1, PRL2, and PRL3 in breast cancer. Br J Cancer 95, 347-54
Wu, X, Zeng, H, Zhang, X, Zhao, Y, Sha, H and Ge, X (2004) Phosphatase of regenerating liver-3 promotes motility and metastasis of mouse melanoma cells. Am J Pathol 164, 2039-54
Zeng, Q, Hong, W and Tan, YH (1998) Mouse PRL2 and PRL3, two potentially prenylated protein tyrosine phosphatases homologous to PRL1. Biochem Biophys Res Commun 244, 421-7
Zhou, H, Gallina, M, Mao, H, Nietlispach, D, Betz, SF, Fetrow, JS and Domaille, PJ (2003) 1H, 13C and 15N resonance assignments and secondary structure of the human protein tyrosine phosphatase, PRL2. J Biomol NMR 27, 397-8
Zhou, J, Wang, S, Lu, J, Li, J and Ding, Y (2009) Over-expression of phosphatase of regenerating liver-3 correlates with tumor progression and poor prognosis in nasopharyngeal carcinoma. Int J Cancer 124, 1879-86
Received 21 September 2011/16 February 2012; accepted 3 May 2012
Published as Cell Biology International Immediate Publication 3 May 2012, doi:10.1042/CBI20110534
© The Author(s) Journal compilation © 2012 International Federation for Cell Biology
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)