Annals of Oncology Advance Access originally published online on July 27, 2006
Annals of Oncology 2006 17(10):1517-1522; doi:10.1093/annonc/mdl159
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© 2006 European Society for Medical Oncology
breast cancer |
Overexpression of phosphatase of regenerating liver-3 in breast cancer: association with a poor clinical outcome
1 Department of Biochemistry and Molecular Biology, Peking University School of Oncology, Beijing Cancer Hospital and Institute, Beijing, China
2 Department of Pathology, Peking University School of Oncology, Beijing Cancer Hospital and Institute, Beijing, China
3 People Hospital of Henan Province, Zhengzhou, HeNan, China
4 Breast Center, Peking University School of Oncology, Beijing Cancer Hospital and Institute, Beijing, China
*Correspondence to: Dr Y. Xie or Dr C. Shou, Peking University School of Oncology, Beijing Cancer Hospital and Institute, 100036 Beijing, P. R. China; Phone: +86-10-88196362 or +86-10-88196766; E-mail: zlxyt2{at}bjmu.edu.cn or cshou{at}vip.sina.com
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Abstract |
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Background: Increasing evidence has suggested that phosphatase of regenerating liver-3 (PRL-3) plays an important role in cancer cell migration, invasion and metastasis. However, the correlation between the PRL-3 expression and clinical outcome in breast cancer has not been investigated.
Patients and methods: Using a PRL-3-specific monoclonal antibody 3B6, PRL-3 expression was assessed by immunohistochemistry in tumor tissues from 382 breast cancer patients with a median follow-up of 65 months.
Results: We found that 34.8% patients expressed a high level of PRL-3 protein in their tumors. Patients with a high level of PRL-3 in the tumor had a worse disease-specific survival (DSS) rate than those with a low level of PRL-3 (74.0% versus 84.9%, P = 0.011), and PRL-3 remained an independent prognostic marker for DSS (HR 1.8, 95% CI 1.1–2.9, P = 0.019) in multivariate analysis. More importantly, in 219 node-negative patients, PRL-3 showed a significant correlation with DSS in univariate analysis (P = 0.014) and retained a borderline significance (HR 2.65, 95% CI 0.92–7.64, P = 0.071) in multivariate analysis.
Conclusions: Our results suggest that PRL-3 may serve as an unfavorable prognostic marker in breast cancer, especially for patients with node-negative diseases. Thus, our findings may provide useful information for individualized therapy in the clinical setting.
Key words: breast cancer, PRL-3, prognosis
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introduction |
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TopAbstractintroductionpatients and methodsresultsdiscussionAcknowledgementsReferences |
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Breast cancer is among the most common diseases in women worldwide and a substantial number of patients eventually die from relapse or metastasis [1, 2]. Although adjuvant treatment may be unnecessary for about 70% of breast cancer patients with node-negative diseases [3], it is difficult to distinguish these patients from the other 30% of patients who may relapse within 10 years after surgery and thus require an adjuvant systemic therapy. Therefore, searching for new prognostic indicators that are able to sort out those patients at high risk of relapse is extremely important.
There has recently been increasing evidence that phosphatase of regenerating liver-3 (PRL-3), also known as PTP4A3, plays an important role in cancer cell migration, invasion and metastasis [4–7]. PRLs constitute a novel class of small tyrosine phosphatases with a C-terminus for prenylation, including three members (PRL-1, PRL-2 and PRL-3) that display approximately 75% homology with each other in an amino acid sequence [8]. The human PRL-3 gene, originally cloned from the human heart [9], is located on chromosome 8q24.3 and encodes a protein of 173 amino acids. Among normal human adult tissues, PRL-3 is expressed in the heart and some striated and smooth muscle cells, with a lower expression in the pancreas [10]. Cells that stably express PRL-3 have exhibited enhanced motility, invasive activity and promoted tumor metastasis in animal models [11–13]. A previous report showed that PRL-3 mRNA was overexpressed in colon cancer and metastatic lesions derived from colorectal cancer [14]. Furthermore, PRL-3 expression was closely associated with lymphatic invasion in gastric carcinomas and with progression in ovarian cancer [15, 16].
Since the three PRL phosphatase members are highly homologous, the polyclonal antibodies against PRL-3 could potentially cross-react with PRL-1 and PRL-2. In order to detect PRL-3 expression at the protein level, we have made the monoclonal antibody 3B6 against PRL-3 and confirmed its specificity with an enzyme-linked immunosorbent assay and Western blotting [17]. We have already applied immunohistochemical staining with this antibody to detect PRL-3 expression in colorectal cancer tissues. Our finding showed that overexpression of PRL-3 was significantly associated with liver metastasis and with a shorter survival time [18]. However, the expression and prognostic value of PRL-3 protein in breast cancer is as yet unknown. In this study, PRL-3 expression was detected by immunohistochemistry in tumor tissues from 382 breast cancer patients with a median follow-up of 65 months; our purpose was to investigate the clinical significance of PRL-3 in this cohort of patients.
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patients and methods |
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study population
A total of 386 patients with operable primary breast cancer were treated at Peking University School of Oncology between 1996 and 1999. Patients older than 75 years were excluded from the study and, therefore, 382 patients were involved in this study. The patients' age ranged from 25 to 75 (with a median of 51.5 years), including 140 premenopausal and 242 postmenopausal patients. The stage of their tumors was classified according to the tumor-node-metastasis classification of the Union Internationale Contre Le Cancer. All patients had received radical or modified radical mastectomy and their axillary lymph nodes had been dissected to at least level I and II. The presence of lymph node metastasis had been determined by histological examination. The majority of patients received adjuvant therapy, including chemotherapy, endocrine therapy, radiotherapy or combined therapy as summarized in Table 1. Follow-up data were available for all patients, with a median follow-up of 65 months. Tumors were considered estrogen-receptor or progesterone-receptor positive if the level of specific hormone binding was equal or greater than 10 fmol/mg of cytosol protein [19, 20]. HER2 expression was determined by immunohistochemistry as described elsewhere [21]. This study was approval by the ethical committee of Peking University School of Oncology.
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specificity analysis of monoclonal antibody 3B6 against PRL-3
To date, there is no commercial antibody specific to PRL-3. We therefore generated a monoclonal antibody 3B6 to PRL-3 in a previous study [17]. Since antibody specificity was critical for this study, 3B6's specificity for PRL-3 was further characterized. We constructed mammalian expression vectors that expressed human PRL-1, PRL-2 or PRL-3 fused with glutathione S-transferase (GST). After DNA sequencing confirmation, the recombinant plasmid DNA was used to transfect COS-7 cells transiently with Lipofectamin 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. At 48 h after transfection, the cells were harvested and lysed in lysis buffer (50 mmol/l Tris-HCl, pH 8.0; 150 mmol/l NaCl; 5 mmol/l EDTA, 1% Nonidet P-40; and 1 mmol/l phenylmethyl-sulfonyl fluoride) on ice. Cell lysates containing equal amounts of protein extract, were separated by 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. Non-specific binding was blocked with 5% non-fat dried milk at 4°C overnight. Next, Western blotting was performed with 3B6 (2.5 µg/ml) followed by a secondary antibody conjugated with horseradish peroxidase. The proteins were detected with the ECL chemiluminescence detection system (Amersham Biosciences, Arlington Heights, IL), according to the manufacturer's instructions. To determine the expression of PRL-GST fusion proteins in transfected COS-7 cells, we used an anti-GST antibody (1 µg/ml) as a control.
immunohistochemistry
For immunohistochemical studies, 4 µm sections were cut from paraffin blocks form 382 cancer tissues and baked at 50–60°C overnight. The paraffin sections were dewaxed with xylene and rehydrated through a graded alcohol series. The endogenous peroxidase activity was then blocked in an absolute methanol solution containing 3% H2O2 for 10 min. After the slides were blocked with 1% bovine serum albumin for 20 min, they were subjected to a 10-min microwave pretreatment in citrate buffer (10 mM) and were then incubated with the 3B6 antibody (2.5 µg/ml) overnight at 4°C in a humidified chamber. EnVision+TM (DOKO, Carpinteria, CA) was used as a secondary antibody. For each step, the slides were washed twice for 5 min with phosphate-buffered saline. Antibody binding was visualized by a standard streptavid in immunoperoxidase reaction, followed by chromagen detection with diaminobenzidine for 10 min and hematoxylin counterstaining. Normal mouse serum was used as a negative control, and the positive slides from our previous study on colon carcinoma were used as a positive control. The staining in the cytoplasm and the cytoplasmic membrane was evaluated. The score for PRL-3 staining was graded as follow: no staining or staining observed in <10% of tumor cells was given a score 0; faint/barely perceptible staining detected in 
10% of tumor cells was scored as 1+; a moderate or strong complete staining observed in 10% of tumor cells was scored as 2+ or 3+, respectively. A score of 0 and 1+ was considered negative, whereas 2+ and 3+ were considered positive. The immunostaining was evaluated independently by three oncologic pathologists without any knowledge of the clinical data.
statistical analysis
A standard 
2 test was performed to assess the association between PRL-3 expression and clinicopathologic characteristics. Disease-specific survival (DSS) time was defined as the time from diagnosis of disease to either death from breast cancer or the date of last contact. Survival curves were estimated using the Kaplan–Meier method and compared with the log rank test. A multivariate analysis was performed by using the Cox proportional hazard regression model (a backward selection) to assess whether a factor was an independent predictor of DSS. Hazard ratios (HRs) with 95% confidence intervals were estimated. A two-tailed P value of less than 0.05 was considered statistically significant. All statistical analyses were performed with SPSS 11.0 software.
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results |
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TopAbstractintroductionpatients and methodsresultsdiscussionAcknowledgementsReferences |
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specificity of monoclonal antibody 3B6 against PRL-3
GST fusion proteins of PRL-1, PRL-2 and PRL-3 were well-expressed in transfected cells. As shown in Figure 1 with the Western blotting, the monoclonal antibody 3B6 reacted with PRL-3 only; even the levels of PRL-1 and PRL-2 proteins were higher than that of PRL-3 protein.
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PRL-3 expression and clinicopathologic characteristics or adjuvant therapy
The expression of PRL-3 in breast tissues was examined by immunohistochemistry. As shown in Figure 2, PRL-3 protein was mainly localized to the cytoplasm and the cytoplasmic membrane of cancer cells. Overexpression of PRL-3 was found in 133 of 382 (34.8%) breast cancer tissues. However, no significant correlation between the PRL-3 expression and clinicopathologic characteristics (e.g. age, tumor size, clinical stage, degree of lymph node involvement, HER2, and ER or PR status) was found in this cohort (Table 1). On the other hand, the adjuvant therapy (e.g. chemotherapy, endocrine therapy, radiotherapy, alone or in combination) was evenly distributed in PRL-3 positive or negative group. No significant difference was found in the two groups (Table 1). Importantly, therefore, the different clinical outcome observed in the two groups was not due to the potential effects of different adjuvant therapies (Table 1).
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PRL-3 expression and breast cancer survival
The 5-year disease-specific survival rate was 81.0% in the whole population. In a univariate analysis, PRL-3 expression was significantly associated with clinical outcome. Patients with a high level of PRL-3 exhibited a lower 5-year disease-specific survival rate than patients with a low level of PRL-3 (74.0% versus 84.9%, P = 0.011, Table 2 and Figure 3A). As expected, axillary lymph node status (P < 0.001), tumor size (P < 0.001), clinical stage (P < 0.001) and HER2 (P = 0.003) were significantly associated with clinical outcome (Table 2). In contrast, patient age and ER or PR status were not significantly associated with disease-specific survival time in this cohort (Table 2). A multivariate analysis showed that PRL-3 expression was an independent prognostic marker with regard to cancer-related survival (HR 1.79, P = 0.019, Table 3), although impact of PRL-3 was less evident than late clinical stage III (HR 2.40, P = 0.008) or more than four lymph nodes metastases (HR 4.68, P < 0.001). The risk of patients with positive PRL-3 status dying from the disease within a specific time was 1.8 times higher than the risk for patients with negative PRL-3 expression (Table 3).
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PRL-3 expression and survival in node-positive or node-negative breast cancer
For further analysis, the patients were divided into two groups according to their nodal status (node-negative or node-positive). In the node-negative subgroup, PRL-3 expression was significantly associated with disease-specific survival in univariate analysis, patients with a low level of PRL-3 expression had a 5-year DSS rate of 95.8% compared with 86.6% among patients with a high level of PRL-3 expression (P = 0.014, Figure 3B). Among the 219 patients with node-negative diseases, 10 out of 74 (13.5%) patients with a high level of PRL-3 expression died of cancer-related causes, but only six out of 145 (4.1%) patients with a low level of PRL-3 expression died within this period of time. In multivariate analysis, PRL-3 showed a borderline significance (95% CI 0.92–7.64, P = 0.071) for correlation with a worse DSS in node-negative patients after adjusting for age, tumor size, ER or PR status, and HER2 status (Table 3). In contrast, no significant correlation between the PRL-3 expression and disease-specific survival was found in patients with node-positive disease (P = 0.142, Figure 3C).
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discussion |
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TopAbstractintroductionpatients and methodsresultsdiscussionAcknowledgementsReferences |
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Although there is limited information on the role of PRL-3 and its molecular targets, it has been suggested to be crucial during the invasion and metastasis of carcinoma cells [22]. For example, it has been shown that stable expression of wild-type PRL-3 dramatically enhanced Chinese hamster ovary cell motility and migration, whereas expression of a catalytically inactive PRL-3 (C104S) mutant greatly reduced the effect on promoting cell migration [13]. Similarly, B16F10 mouse melanoma cells stably expressing PRL-3 displayed a fibroblast-like appearance and showed much higher migratory ability than its lowly metastatic parental cell line. PRL-3 expression also facilitated lung and liver metastasis of B16F10 cells in an animal model [11]. A recent study suggested that PRL-3 was the only gene consistently overexpressed in 18 colorectal cancer liver metastases examined [5]. Likewise, Bardelli et al. [6] also reported that PRL-3 mRNA expression was elevated in nearly all metastatic lesions derived from colorectal cancers, regardless of the site metastasis. Taken together, these studies imply that PRL-3 plays a crucial role in tumor cell metastasis and invasion.
In the present study, we assessed PRL-3 protein expression in 382 breast cancer tissues by using immunohistochemistry. We found that PRL-3 expression was significantly associated with clinical outcome; patients with PRL-3-negative tumors had substantially longer DSS than did patients with PRL-3-positive tumors. Furthermore, multivariate analysis showed that positive PRL-3 expression was an independent marker for DSS in the entire population after adjusting for other prognostic factors. We previously found that PRL-3 expression is significantly associated with liver metastases and a shorter survival time in a subset of 88 patients with colorectal cancer [18]. Thus, our present results are consistent with the previous findings.
In the node-negative subgroup, PRL-3 showed a significant correlation with worse clinical outcome in univariate analysis, and retained a borderline significance for DSS after adjusting for age, tumor size, ER or PR status, and HER2 status, whereas in node-positive patients, PRL-3 seemed to have no influence. Interestingly, we also found the PRL-3 expression was neither associated with the tumor size nor with the degree of lymph node involvement, suggesting that PRL-3 expression was independent of lymph node metastases. Thus, we raise a hypothesis that PRL-3 may contribute to breast cancer metastasis via lymph-node independent pathway. Our findings may have clinical implications: the evidence that approximately 30% node-negative breast cancer patients eventually succumb to the disease due to distant metastases suggests that PRL-3 may serve as a potential prognostic marker for node-negative breast cancer patients.
Parker et al. [23] found that PRL-3 mRNA appeared to be expressed predominately in the vasculature of invasive breast cancer by in situ hybridization. But PRL-3 protein was predominately located in the cytoplasm and cytoplasmic membrane of cancer cells in our study, consistent with previous reports [6, 15, 18]. We also found the weak expression of PRL-3 in endothelial cells in few cancer tissues (figure not shown). The distribution pattern of PRL-3 in the cell may correlate with the metastatic ability of the tumor cells. A previous study showed that cells expressing PRL-3 were enriched in several membrane processes including protrusions, ruffles and some vacuolar-like membrane extensions, all of which have been reported to play a role in invasion and cell movement [24, 25]. PRL-3 may also induce dephosphorylation of target substrates at the cell membrane and modulate the organization of the plasma membrane in such a way to promote cell motility and loss of adhesion [9]. We recently found that PRL-3 activates the mitogen-associated protein kinase pathway, which is mediated by its association with a protein located at the cell membrane that is involved in cell migration and invasion [26].
In conclusion, our study suggests that PRL-3 expression may serve as an unfavorable prognostic factor in breast cancer patients, particularly for node-negative patients. Detection of PRL-3 expression may provide useful information to discriminate the node-negative patients who may have a high risk of relapse, and allow the low-risk patients to avoid harmful chemotherapy. Nevertheless, other independent studies are needed to confirm our present findings.
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This work was supported by grants from the Development Foundation of Capital Medical Science (No. 2002–2023, to Dr C. Shou) and Chinese National Basic Research Program (No. 2004CB518701, to Dr C. Shou). We thank Dr Jiyou Li and Dr Jingsheng He for the contribution of the tumor samples. We also thank Dr Hongxia Li for help with the statistical analysis.
Received for publication April 26, 2006. Revision received May 29, 2006. Accepted for publication May 30, 2006.
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