ANTICANCER RESEARCH 29: 3913-3916 (2009)

PTP4A3 Expression Increases Strongly in Lymph Node Metastases from Colorectal Carcinoma

KATARZYNA GUZIŃSKA-USTYMOWICZ, ANNA PRYCZYNICZ and ANDRZEJ KEMONA

Department of General Pathomorphology, Medical University of Białystok, Białystok, Poland

Abstract. Background: Numerous studies have proven that disseminate from primary tumors to lymph nodes as well as protein tyrosine phosphatase type IV A member 3 (PTP4A3) to the nearest and distant tissues and organs is an essential plays a significant role in local lymph node metastasis from feature of malignant neoplasms and the main cause of colorectal carcinoma. The aim of the current study was to therapeutic failures. Formation of metastases is a complex assess PTP4A3 expression in colorectal carcinomas and and multistage process involving proteolysis, mobility and lymph nodes in correlation with chosen anatomoclinical migration of cells, proliferation and angiogenesis. Cancer parameters. Patients and Methods: Forty-nine patients were cells released from the primary tumor have to invade the analyzed with respect to age, tumor location, pTN stage and adjacent tissues, permeate lymphatic or blood vessels, local lymph node involvement. The level of PTP4A3 migrate through the vascular walls to the adjacent tissues, expression was determined immunohistochemically using where they eventually settle, proliferate and induce mouse monoclonal anti- PTP4A3 antibody (monoclonal angiogenesis, forming metastatic foci. Early detection of antibody 3B6, anti-human PTP4A3 from Attogen Biomedical metastases offers a good chance of a cure. A recent study Research, USA). Results: Positive reaction for PTP4A3 in the indicated that the protein formerly known as phosphatase of main tumor mass was observed in 9/49 (18.4% ) cases, regenerating liver-3 (PRL-3) newly named protein tyrosine whereas in metastatic lymph nodes in 22/24 (91.6% ) cases. phosphatase type IV A member 3 (PTP4A3) could be a Statistical analysis showed no correlation of PTP4A3 marker for metastasis (1). expression in the main tumor mass with such clinical The PTP family comprises a large group of enzymes parameters as age, location, histological type or lymph node taking part in one of the most important reactions in the cell, involvement. Moreover the expression of PTP4A3 in the main i.e. dephosphorylation of tyrosine residues that are involved tumor mass was not associated with the expression of this in activation or inactivation of enzymes. In this way, they protein in lymph node metastases. However, there was a regulate a number of cellular processes, both physiological statistically significant correlation between lymph node and pathological, including: cell growth, differentiation, involvement and PTP4A3 expression in lymph node cycle or neoplastic transformation. Non-classic metastases. Conclusion: The role of the PTP4A3 has not yet representatives of this family include three closely related been fully elucidated. Our finding indicate PTP4A3 is phosphatases (PTP4A1-PTP4A3) with a unique COOH strongly expressed in colorectal carcinoma metastases of the terminal group. All these proteins have at least 75% of their lymph nodes although the significance of this is unclear. amino acid sequences in common. PTP4A1 is localized primarily in the brain and muscles, PTP4A2 in skeletal Colorectal carcinoma is one of the most common causes of muscles and PTP4A3 in cardiac muscle and skeletal muscles death due to malignancy, its incidence showing a growing (2). PTP4A3 protein has an active site with the signature tendency. Tumor stage is the major prognostic factor and motif CX5R, where Cys104 is an enzymatic nucleophile and colorectal carcinoma frequently metastasizes to local lymph Arg110 binds to phosphate groups on phosphotyrosine (3). nodes, liver and lungs. The ability of cancer cells to The PTP4A3 molecule contains C-terminal consensus sequence for prenylation, and it is localized in membranes and intracellular structures when prenylated and in the nucleus when it is not. The role of PTP4A3 is not well Correspondence to: Katarzyna Guzińska-Ustymowicz, MD, known. Matter et al. (4) have suggested that PTP4A3 may Department of General Pathomorphology, Medical University of play a role in the regulation of intracellular calcium Białystok, ul. Waszyngtona 13, 15-269 Bialystok, Poland. Tel: +48 857485942, Fax: +48 857485996, e-mail: [email protected] transmitters induced by angiotensin II. Wu et al. (5) discovered the presence of PTP4A3 in cellular membrane Key Words: PTP4A3, colorectal carcinoma. components in the mitotic phase specifically in metaphase.

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This location may suggest that PTP4A3 is engaged in cell Table I. PTP4A3 expression in the main tumor mass and in metastatic cycle regulation. The protein also plays a significant role in lymph nodes in association with anatomoclinical parameters. the induction of angiogenesis by recruiting endothelial cells Parameters PTP4A3 from the circulating blood and in the formation of microcirculation (6). However, abnormal regulation of Tumor Lymph nodes tyrosine phosphorylation and dephosphorylation may also lead to neoplastic formation. PTP4A3 protein expression has –+ p-Value – + p-Value been detected in cancer of the ovaries (7), breast (8), Age stomach (9) and large intestine (1). <60 years 7 3 0.297 6 4 0.631 The aim of the present study was to assess the expression ≥60 years 33 6 20 19 of PTP4A3 in colorectal carcinomas and in lymph nodes in Localization correlation with chosen anatomoclinical parameters. Rectum 22 7 0.217 14 15 0.430 Colon 18 2 12 8 Histological type of cancer Patients and Methods Adenocarcinoma 35 9 0.272 24 20 0.547 Adenocarcinoma gelatinosum 5 0 2 3 The study was conducted in a group of 49 patients treated surgically Lymph node metastasis due to colorectal carcinoma in the Department of Surgery, the J. Absent 20 5 0.769 24 1 <0.0001 Śniadecki Hospital in Białystok. Sections, 4 μm thick, were cut Present 20 4 2 22 from paraffin blocks and stained with hematoxylin-eosin (H+E). Distant metastasis The routine histopathological assessment was performed for Absent 35 9 0.272 24 20 0.547 histological type, malignancy grade (G), infiltration depth and the Present 5 0 2 3 presence of lymph node metastases. PRL-3 in lymph nodes – 21 5 0.120 - - - Immunohistochemical analysis. Formalin-fixed and paraffin- +194 embedded tissue specimens were cut on a microtome into 4 μm p<0.05 was considered significant. sections. The sections were deparaffinized in xylene and hydrated in alcohol. To visualize the antigen, the sections were heated in a microwave oven for 15 min in a citrate buffer (pH 6.0). They were incubated with 0.5% hydrogen peroxide solution in methanol in order to block endogenous peroxidase. Incubation was then parameters as age, location, histological type, the presence performed with mouse monoclonal antibody against human PTP4A3 of metastases in lymph nodes and distant metastases. The (monoclonal antibody 3B6; Attogen Biomedical Research, USA) relationship between PTP4A3 expression in the main tumor over night at 4˚C. The secondary reaction was carried out using mass was not correlated with PTP4A3 expression in lymph biotinylated anti-mouse antibody conjugated with horseradish nodes. Nor was PTP4A3 positivity in lymph nodes correlated peroxidase (LSAB2, DAKO, Poland). A colour reaction for with other factors. The relationship between the presence of peroxidase was developed with the chromogen diaminobenzidine (DAKO). Protein expression was determined using a lymph node involvement and PTP4A3 expression in the semiquantitative method and assessed as positive (reaction visible lymph nodes was, however, statistically significant in >5% of tumour cells) or negative (lack of reaction, or reaction (p<0.0001) (Table I). present in <5% of cells). Reactions were assessed in at least 500 cancer cells in each tissue specimen under a light microscope Discussion (×400). Statistical analysis was conducted using Fisher’s test and χ2 The current study objective was to analyze the role of analysis. A p-value <0.05 was considered statistically significant. PTP4A3 expression in colorectal cancer in correlation with Results the presence of lymph node involvement. The expression of PTP4A3 in the main tumor mass was significantly lower than The study group consisted of 49 patients with colorectal that in the lymph node metastases. Saha et al. (1), who carcinoma pT3G2. Twenty-four patients (49% ) had lymph conducted a molecular study on PTP4A3 gene expression, node metastases and four (8% ) distant metastases. noticed that it was high in 18/18 metastases, whereas low in Cytoplasmic PTP4A3 expression in the main mass of a non-metastasizing carcinomas and in normal epithelial tumor and no reaction in normal mucosa has been presented tissue. Interestingly, in 3/12 metastases they found duplicate in Figure 1. Positive reaction for PTP4A3 protein in the main copies of the PTP4A3 gene localized in a small amplicon at tumor mass was observed in 9/49 (18.4% ) cases, whereas in chromosome 8q24.3, which suggests the significance of the metastatic lymph nodes, in 22/24 (91.6% ) cases. Statistical PTP4A3 gene in colorectal carcinoma. In addition, Buffart et analysis did not show any correlation between PTP4A3 al. (10) compared the number of PTP4A3 gene copies in the expression in the main mass of tumor and such clinical primary tumor of colorectal carcinoma with and without

3914 Guzińska-Ustymowicz et al: PTP4A3 Expression in Colorectal Cancer

Figure 1. Immunohistochemical staining. A, Lack of staining for PTP4A3 in normal mucosa. B, Colorectal cancer tissue showing cytoplasmic staining for PTP4A3. Original magnification, ×400.

metastases to the liver. These observations seem to suggest in lymph node metastases was high in 22/24 cases, but the that metastasis may be associated with the genetic profile of protein expression in the main tumor mass did not correlate the primary tumor and that amplification of the PTP4A3 with the presence of metastases, in contrast to the finding of gene is a mechanism leading to protein over expression. Wang et al. (14). They also found high expression of PTP4A3 in the vascular Although our findings show no correlation between system of the tumor irrespective of the source of the PTP4A3 expression and colorectal cancer metastasis per se, neoplasm. Bardelli et al. (11) observed high PTP4A3 mRNA its high expression in lymph node metastases is of expression in colorectal cancer metastases to other organs (in significance. PTP4A3 involvement in colorectal cancer 10/11 metastases to the liver, 6/7 to the lungs, 4/4 to the metastasis needs to be further investigated. brain and in 3/3 to the anus), whereas there was no or only low expression of the protein in metastases to these organs Acknowledgements from other types of carcinoma (pancreatic, gastric, esophageal) and in non-metastasizing colorectal carcinoma. We thank Dr. Jerzy Hapanowicz, Department of Surgery Hospital Moreover, they discovered that the expression of PTP4A3 in Białystok for cooperation. was higher in over 95% of metastases as compared to the increased number of PTP4A3 gene copies in colorectal References cancer (1), thus showing that PTP4A3 gene amplification is 1 Saha S, Bardelli A, Buckhaults P, Velculescu VE, Rago C, St not the major cause of the protein overexpression. It was also Croix B, Romans KE, Choti MA, Lengauer C, Kinzler KW and demonstrated that PTP4A3 overexpression was not due to the Vogelstein B: A phosphatase associated with metastasis of presence of neoplastic metastatic cells in the liver. Kato et colorectal cancer. Science 294: 1343-1346, 2001. al. (12) observed similar relations in PTP4A3 protein 2 Bessette DC, Qiu D and Pallen CJ: PRL PTPs: mediators and expression in colorectal carcinomas and their metastases. markers of cancer progression. Cancer Metastasis Rev 27: 231- They also showed that PTP4A3 overexpression is associated 252, 2008. with the mobility of neoplastic cells but had no effect on 3 Kim KA, Song JS, Jee J, Sheen MR, Lee C, Lee TG, Ro S, Cho JM, Lee W, Yamazaki T, Jeon YH and Cheong C: Structure of their proliferation. human PRL-3, the phosphatase associated with cancer In an immunohistochemical study concerning PTP4A3 metastasis. FEBS Lett 565: 181-187, 2004. expression, Peng et al. (13) observed high overexpression of 4 Matter WF, Estridge T, Zhang C, Belagaje R, Stancato L, Dixon this protein in colorectal cancer metastasis to local lymph J, Johnson B, Bloem L, Pickard T, Donaghue M, Acton S, nodes (53.7% cases) and also in liver metastases (66.7% of Jeyaseelan R, Kadambi V and Vlahos CJ: Role of PRL-3, a cases), whereas there was low protein expression in non- human muscle-specific tyrosine phosphatase, in angiotensin-II metastasizing tumor (23.9% of cases) as well as in normal signaling. Biochem Biophys Res Commun 283: 1061-1068, 2001. colorectal epithelium (7.1% of cases). The expression of 5 Wu X, Zeng H, Zhang X, Zhao Y, Sha H, Ge X, Zhang M, Gao PTP4A3 protein in the main tumor was found to correlate X and Xu Q: Phosphatase of regenerating liver-3 promotes with lymph node involvement, but not with the presence of motility and metastasis of mouse melanoma cells. Am J Pathol distant metastases. In our study, the expression of PTP4A3 164: 2039-2054, 2004.

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6 Guo K, Li J, Wang H, Osato M, Tang JP, Quah SY, Gan BQ and 12 Kato H, Semba S, Miskad UA, Seo Y, Kasuga M and Yokozaki Zeng Q: PRL-3 initiates tumor angiogenesis by recruiting H: High expression of PRL-3 promotes cancer cell motility and endothelial cells in vitro and in vivo. Cancer Res 66: 9625-9635, liver metastasis in huma colorectal cancer: a predictive molecular 2006. marker of metachronous liver and lung metastases. Clin Cancer 7 Polato F, Codegoni A, Fruscio R, Perego P, Mangioni C, Saha Res 10: 7318-7328, 2004. S, Bardelli A and Broggini M: PRL-3 phosphatase is implicated 13 Peng L, Ning J, Meng L and Shou C: The association of the in ovarian cancer growth. Clin Cancer Res 11: 6835-6839, 2005. expression level of protein tyrosine phosphatase PRL-3 protein 8 Wang L, Peng L, Dong B, Kong L, Meng L, Yan L, Xie Y and with liver metastasis and prognosis of patients with colorectal Shou C: Overexpression of phosphatase of regenerating liver-3 cancer. J Cancer Res Clin Oncol 130: 521-526, 2004. in breast cancer: association with a poor clinical outcome. Ann 14 Wang Y, Li ZF, He J, Li YL, Zhu GB, Zhang LH and Li YL: Oncol 17: 1517-1522, 2006. Expression of the human phosphatases of regenerating liver 9 Miskad UA, Semba S, Kato H and Yokozaki H: Expression of (PRLs) in colonic adenocarcinoma and its correlation with PRL-3 phosphatase in human gastric carcinoma: close lymph node metastasis. Int J Colorectal Dis 22: 1179-1184, correlation with invasion and metastasis. Pathobiology 71: 176- 2007. 184, 2004. 10 Buffart TE, Coffa J, Hermsen MA, Carvalho B, van der Sijp JR, Ylstra B, Pals G, Schouten JP and Meijer GA: DNA copy number changes at 8q11-24 in metastasized colorectal cancer. Cell Oncol 27: 57-65, 2005. 11 Bardelli A, Saha S, Sager JA, Romans KE, Xin B, Markowitz SD, Lengauer C, Velculescu VE, Kinzler KW and Vogelstein B: Received May 11, 2009 PRL-3 expression in metastatic cancers. Clin Cancer Res 9: Revised August 30, 2009 5607-5615, 2003. Accepted September 1, 2009

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