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Home , PTEN (gene), PTPN13, PTPN14, PTPN21, PTPN3, PTPN4

ANTICANCER RESEARCH 26: 1643-1650 (2006)

PTPN3 and PTPN4 Tyrosine Expression in Human Gastric Adenocarcinoma

CHEW-WUN WU1, JEN-HAO CHEN1, HWA-LI KAO2, ANNA F.-Y. LI2, CHUN-HUN LAI5, CHIN-WEN CHI3 and WEN-CHANG LIN4,5

Departments of 1Surgery, 2Pathology and 3Medical Research and Education, Taipei Veterans General Hospital, Taipei 112, Taiwan; 4Institute of Bioinformatics, School of Medicine, National Yang-Ming University, Taipei 112, Taiwan; 5Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, R.O.C.

Abstract. Background: Degenerated PCR primers, designed related signaling molecules (1). Amongst according to the consensus tyrosine phosphatase catalytic motifs, events, modulated by protein kinases were used in order to amplify expressed protein-tyrosine and is an important post-translational phosphatase molecules from human gastric -derived cells. modification event for a variety of cells. Such phosphorylation From such profiles, more than twenty different types of tyrosine plays a critical function in signal transduction, , phosphatase were identified from gastric cancer tissue. A non- differentiation and oncogenesis (2-4). Protein-tyrosine tyrosine phosphatase, PTPN4, was found to be expressed phosphatases attenuate the growth signals generated by in a tumor-tissue profile with only low frequency. The most protein-tyrosine kinases by catalyzing the tyrosine closely-related to tyrosine phosphatase, PTPN3, has been step for their substrate (5, 6), shown to be mutated in cases of human , but although the understanding of the cellular functions of the its expression is cases of gastric cancer is not known. Materials protein-tyrosine phosphatases generally lags behind that of the and Methods: The mRNA expression of PTPN3 and PTPN4 by tyrosine kinases. Some protein-tyrosine phosphatases have RT-PCR was investigated, and the protein expression status of been implicated in oncogenic processes, and occur in human PTPN3 was examined, using immunohistochemistry, to elucidate cancer cells as (7, 8) or as tumor-suppressor , clinicopathological associations of the PTPN3 and PTPN4 such as PTEN tyrosine phosphatase (9, 10). For human gastric family within human stomach cancer. Results: PTPN3 and , it has been previously reported that various protein- PTPN4 were expressed in all gastric cancer cell lines and clinical tyrosine phosphatases can serve as Helicobacter pylori cancer tissue specimens examined. Following the examination of cytotoxin receptors (11-13). Following the completion of the 92 gastric cancer patients’ pathological specimens, PTPN3 project, systematic examination of showed no statistical significance with respect to the expressions by microarray (14) and other techniques have patients’ survival. A statistically significant correlation between helped researchers to learn more about the gene regulation PTPN3 staining and the differentiation status of gastric cancer and protein-protein interaction pathways. Thus, the tissue was, however, observed. Conclusion: This finding indicates identification of critical molecules involved in complex that both PTPN3 and PTPN4 are expressed within human gastric interaction pathways, such as those involving the tyrosine cancer cells and that PTPN3 seems to play an important role in kinases and phosphatases, is feasible (15). gastric cancer differentiation and the progression of malignancy. There are, however, drawbacks to the use of a cDNA microarray approach. In addition to concerns regarding data Signal transduction pathways have been implicated in human reproducibility and accuracy, cross hybridization between oncogenesis processes, especially those incorporating growth- EST clones spotted in the array as well as cross-hybridization between paralled genes expressed in cells, could compromise entire microarray experiments (16). Low expression level gene transcripts, such as protein-tyrosine kinases and Correspondence to: Wen-chang Lin, Institute of Biomedical Sciences, phosphatases, might not be successfully identified in Academia Sinica, Taipei 115, Taiwan, R.O.C. Tel: (886) 2-2652- microarray with current the labelling chemicals and 3967, Fax: (886) 2-27827654, e-mail: [email protected] techniques, although these genes could play key roles in some Key Words: Protein tyrosine phosphatase, immunohistochemistry, cellular functions (16). For many laboratories, it is preferable gastric cancer. to examine genes within a biochemical pathway and/or groups

0250-7005/2006 $2.00+.40 1643 ANTICANCER RESEARCH 26: 1643-1650 (2006) of genes with similar functions under differential cellular RNA, and oligo (dT)15 and MMLV reverse transcriptase provided regulations (i.e., gene families). Recently, a systematic by Promega (Madison, WI, USA). The resulting cDNA specimens examination of the protein-tyrosine phosphatase family in the were subjected to PCR reaction with gene-specific primers. RT-PCR was carried out in a 25 Ìl total volume containing 1.5 mM MgCl , context of human colorectal cancers was reported (15), in 2 0.8 ÌM of each primer, 200 ÌM of each dNTP, 20 ng cDNA and one which a total of 87 protein-tyrosine phosphatases from unit of Takara Taq polymerase. The reactions initially took place at amongst 18 colorectal cancer-derived specimens were 94ÆC and continued for 10 min, followed by 38 cycles at 94ÆC for 30 screened at their coding sequences. Only 6 genes featuring sec, 60ÆC for 30 sec and 72ÆC for one min. The final extension phase somatic were identified, including 3 receptor was conducted at 72ÆC for a period of 10 min. For the control tyrosine phosphatases (PTPRF, PTPRG, PTPRT) and 3 non- housekeeping gene (GAPDH), the PCR cycle was reduced to 23 receptor tyrosine phosphatases (PTPN3, PTPN13, PTPN14), cycles. The primer sets for the kinases studied were as follows: from which the mutated protein-tyrosine phosphatase PTPRT Forward Reverse was subjected to further biochemical studies in order to clarify its roles in the transformation process (15). GAPDH 5’TGGTATCGTG- 5’AGTGGGTGTC- Of the 6 above-mentioned protein-tyrosine phosphatase GAAGGACTCA3’ GCTGTTGAAG3’ genes, PTPN3, also known as PTP-H1, was first isolated from PTPN3 5’TGTAAACTATGT- 5’AGCTCCACCTA- HeLa cells (17), and the N-terminal segment of this gene GAGGTCTCTG3’ GAAGCACAGAA3’ was found to display homology to the domains in the -associated protein bands 4.1, ezrin and talin PTPN4 5’AGATCTGGGAT- 5’AGAGCTGACTC- (18). To our knowledge, there have been several reports ATGTGTTGGAA3’ CTGATGTTTAC3’ pertaining to the expression of PTPN3 for gastrointestinal tract-related human cancers (19-21), including esophageal The expected sizes of the PCR products were: 371 bp for GAPDH, 720 bp for PTPN3 and 402 bp for PTPN4. The final cancer, colorectal cancer and hepatocellular carcinoma. products were analyzed in a 1% agarose gel, visualized by ethidium- PTPN3 and its closest homolog, PTPN4, are 2 human bromide staining and recorded with an Alpha Innotech Co. IS-500 protein-tyrosine phosphatases featuring gel-documentation system, with the level of all gene expressions to some cytoskeletal proteins. PTPN4, also known as being determined by the intensity of the signal for the final product. PTPMEG, was first isolated from a megakaryoblastic cell line The ratio of PTPN3/GAPDH to PTPN4/GAPDH expression for each (22). PTPN4 was found to be able to block soft agar colony sample was used as an index of PTPN3 and PTPN4 expressions. formation of transfected COS-7 cells and elicit inhibitory Immunohistochemical staining of protein-tyrosine phosphatase effects on the growth of transfected COS-7 cells (23). PTPN3. The relative resource of protein-tyrosine phosphatase is In our previous protein-tyrosine phosphatase expression less than that of protein-tyrosine kinases, and the number of profile analysis, PTPN4 was found to be expressed within protein-tyrosine phosphatase-specific antibodies available for gastric cancer mucosal tissue, but not in normal gastric immunohistochemical purposes is also somewhat limited. Since mucosal tissue. Here, the expressions of PTPN3 and PTPN4 only the PTPN3-specific antibody was available for in human gastric cancer are further investigated. immunohistochemistry, and given that several previous studies have reported the relative significance of PTPN3 in digestive tract tumors (19, 21), the protein expression status of PTPN3 for human Materials and Methods gastric cancer tissue was the focus of further examination. Human gastric adenocarcinoma tissue was obtained from Gastric cancer cell lines, surgical specimens and mRNA extraction. approximately 90 patients who underwent gastrectomy at the Human gastric cancer cell lines, namely HR, AZ521, AGS, KATO Department of Surgery, Taipei Veterans General Hospital. None III, NUGC, TSGH and SC-M1, were used in this study (24). The of the patients had undergone any chemotherapy or radiotherapy cells were cultured in either RPMI 1640 or DMEM culture prior to their surgery. The maximum time between stomach medium, as appropriate, supplemented with 10% fetal calf serum, removal and the quenching of the removed tissue was 1 h. Tissue 2 mM glutamine, 100 U/ml penicillin and 100 Ìg/ml streptomycin in blocks were fixed overnight at 4ÆC with 4% neutral-buffered 5% CO2/95% air at 37ÆC. Gastric cancer mucosal tissues and their paraformaldehyde solution, dehydrated, cleared with Hemo-De corresponding normal gastric mucosal tissues were obtained from solution (Fisher, Pittsburgh, PA, USA; ingredients: d-limonene, patients who had undergone gastrectomy at Taipei Veterans butylated hydroxanisole) and then embedded in paraffin wax. Five- General Hospital, Taiwan. Informed consent was obtained from all Ìm-thick sections were used for staining. The gastric the study-participating patients. The specimens were immediately adenocarcinomas were histologically divided into intestinal and frozen in liquid nitrogen after resectioning. Total RNA was diffuse classes according to Lauren’s criteria, and both types existed extracted from gastric cancer and normal mucosal tissue by direct either alone or in combination with each other in the tumor tissues. quanidine isothiocyanate lysis and a cesium-chloride gradient- Protein-tyrosine phosphatase (PTPN3) within gastric cancer tissue separation method, as described previously (25). was localized using the avidin-biotin-peroxidase complex technique (25). All histochemistry reagents were obtained from Vector RT-PCR expression analysis of protein-tyrosine phosphatase PTPN3 Laboratories (Burlingame, CA, USA). PTPN3/PTP-H1 (N-19)/sc- and PTPN4. Reverse transcription was conducted using 2 Ìg total 9789 is an affinity-purified goat polyclonal antibody raised against

1644 Wun et al: PTPN3 and PTPN4 Expression in Human Gastric Cancer a peptide corresponding to the amino terminus of PTP-H1 of this protein-tyrosine phosphatase profile. Subsequent to human origin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). examining the expression frequency of each protein-tyrosine Briefly, the rehydrated tissue sections were first treated with phosphatase, it appeared that a greater number of non- microwave irradiation in a sodium citrate buffer solution (0.06 mol/l, receptor-type protein-tyrosine phosphatases were expressed pH=6), and then with peroxide and normal rabbit serum in order to remove endogenous peroxidase activity and to reduce non-specific within normal gastric mucosa, and a greater variety of background staining, respectively. The tissue sections were then receptor protein-tyrosine phosphatases were expressed incubated overnight with goat anti-human PTPN3 antiserum at room within the cancerous tissue. The only non-receptor protein- temperature in a moist chamber. The tissue sections were tyrosine phosphatase expressed in gastric cancer tissue, but subsequently treated with biotin-labelled anti-goat immunoglobulin not within normal gastric mucosa, was PTPN4 for this G antibody (50 Ìl) in 10 ml of phosphate-buffered saline (PBS), expression profile. We decided to further explore the followed by treatment with the avidin-biotin-peroxidase complex. expression of PTPN4 and its closest homologous gene Fresh avidin-biotin-peroxidase complex was made by incubating 10 Ìl of avidin with 10 Ìl of biotin-peroxidase in PBS. Avidin-biotin- (PTPN3) in human gastric cancer samples. peroxidase complex staining for negative controls was carried out by PTPN4 is a 926 amino acid non-receptor phosphatase, omission of the primary antiserum or replacement of the primary while PTPN3 is 913 amino acid residues long (17,22). As antiserum by non-immune goat normal serum. The tissue section illustrated in Figure 1A, the motifs are adjacent to that utilized for avidin-biotin-peroxidase complex staining similar for PTPN3 and PTPN4, both of which include band was stained with hematoxylin and eosin for comparison purposes. 4.1 homolog domains, FERM_C motif, PDZ domain and All staining procedures were standardized and controlled with the tyrosine phosphatase catalytic domain. At the amino an auto-immunostaining workstation (Leica ST5050; Leica, Deerfield, IL, USA). Stained tissue sections were independently acid sequence level, 51% identities (488/951) and 67% viewed and recorded by 2 researchers, and reconfirmed in the case similarities (638/951) were noted between PTPN3 and of discrepancy. For assessing a PTPN3 immunoreactivity, the ratio PTPN4 phosphatases. An RT-PCR approach was first used of the positively-stained epithelial cells to total epithelial cells was to examine the expression status of these 2 phosphatases for used. A minus symbol (–) was used if there were no positively- gastric cancer cell lines and clinical cancer samples. All stained cells present; (+) indicated that >1% but <25% of cells samples examined were positive for PTPN3 and PTPN4 stained positively; (++) indicated that between 25 and 75% of cells expression (Figure 1B). It would appear that the expression stained positively for PTP immunoreactivity; (+++) indicated >75% of cells were positively-stained. However, only weakly- levels of PTPN3 and PTPN4 were low, since the PCR stained cells were (occasionally) observed in the matching normal products were only detectable at high PCR cycle numbers (adjacent non-tumor tissue) gastric epithelium. (38 cycles), when compared with the outcome for the internal control housekeeping gene GAPDH (23 cycles). Clinicopathological and statistical analysis. All clinicopathological This rather low expression level correlated reasonably with information was collected and expressed as a mean±SD, and the our own protein-tyrosine phosphatase profiling result correlations between various disease parameters were analyzed by the Student’s t-test and/or the Chi-square test. The difference was (PTPN4 was expressed at 1.4% in the gastric cancer tumor considered to be significant when the p value was less than 0.05. profile, although it was not expressed in the normal gastric mucosa profile). It is interesting to note that, for clinical Results gastric cancer tissue, the average expression level of PTPN4 appears to be greater than that for PTPN3 (Figure 1B). We have previously reported a RT-PCR-based tyrosine Following the quantitation of PCR product intensity and its kinase profiling approach to studying human gastric cancer, normalization with the housekeeping GAPDH gene, it which revealed the expression of more than 30 different would appear that the mean expression ratio of tyrosine kinases and a few dual kinases in various human PTPN3/PTPN4 for 7 gastric cancer cell lines was almost gastric cancer tissues (25-27). These studies identified a double that for the 5 gastric cancer tissue samples number of differentially-expressed tyrosine kinases that had investigated. As a consequence of the mixture of different previously gone unrecognized from within human gastric cell types in clinical gastric cancer tissue samples, it would cancer cells, including tie-1. Recently, we applied a similar also appear likely that the PTPN3 expression is elevated for protein-tyrosine phosphatase profiling approach to a pair of cancer populations, as indicated by the homogeneous cell human gastric cancer samples and identified the presence line data. In addition, since the PTPN3 expression level was of 22 different types of phosphatases (Wu et al., submitted). elevated for cases of esophageal cancer, colorectal cancer Seven non-receptor-type protein-tyrosine phosphatases and hepatocellular carcinoma, the expression of this non- (PTPN4, PTPN6, PTPN7, PTPN9, PTPN11, PTPN14 and receptor tyrosine phosphatase for cases of human stomach PTPN21) and 15 receptor-type protein-tyrosine cancer should be examined. phosphatases (PTPRA, PTPRB, PTPRC, PTPRCAP, PTPRD, Following the immunohistochemical staining of the PTPN3 PTPRE, PTPRF, PTPRG, PTPRH, PTPRJ, PTPRKappa, protein in pathological sections deriving from the 92 gastric PRPRN, PTPRO, PTPRS and PTPRZ) were expressed in cancer patients, the clinicopathological relevance of gastric

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Figure 1. PTPN3 and PTPN4 gene protein motif organization and their expression in human gastric cancers. (A) With high homology, PTPN3 and PTPN4 share similar protein domain motifs and organization. B41: Band 4.1 homologs; FERM_C: the third structural domain within the FERM domain; PDZ: PDZ signaling domain; and PTPc: protein-tyrosine phosphatase catalytic domain. The RT-PCR primers’ positions are marked as F: forward primer and R: reserve primer. (B) Expression of PTPN3 and PTPN4 for 7 human gastric cancer cell lines, AGS, AZ521, HR, Kato III, NUGC, SC-M1 and TSGH (left panel) and 5 gastric cancer tissues, G38T, G39T, G40T, G41T and G42T (right panel). Total RNA samples were prepared from each cell line and tissue as described in the Materials and Methods section. RT-PCR reactions, using primers specific for PTPN3 and PTPN4 genes, were performed with 38 PCR cycles. The GAPDH gene served as an internal PCR reaction control. The final products were analyzed in a 1% agarose gel.

cancer progression was correlated with the PTPN3 expression Discussion status for these samples. The immunohistochemical results of PTPN3 antibody are provided in Figure 2. Positive Previously, we have been able to successfully identify kinase immunoreactivity existed mostly in the cytoplasm regions of genes that had not been recognized as being expressed in cancer cells as expected. Color intensity was not considered a gastric cancers, and have validated their existence in human valid parameter for assessment of immunohistochemical cancer cells by subsequent analysis. Some of these genes reactions occurring within the stained cells because of the were found to be important for the growth and progression difficulties associated with objective and quantitative of tumor cells and showed strong correlations with measurement in this system. PTPN3 expression was observed prognosis (1). We have demonstrated that the expression of in 53% of the samples examined. The clinicopathological tie-1 and mkk4 kinase protein within gastric-cancer tissues features investigated included age, sex, tumor size, tumor is correlated with the patients’ prognostic outcome (25, 27). location, gross tumor type, stromal reaction pattern, depth of In order to elucidate the complete regulatory mechanisms cancer invasion, lymphatic invasion, vessel invasion, lymph-node in the signal transduction pathways, a general and metastasis, liver metastasis, peritoneal dissemination, Lauren’s comprehensive tyrosine kinase/ tyrosine-phosphatase tumor histological classification rating and differentiation grade. expression profile in cancer cells would clearly benefit Our data indicated no statistical significance with respect to the oncologists. Recently, a systematic examination of tyrosine patients’ survival as regards PTPN3 expression. From Table I, phosphatase mutations for human colorectal cancer has expression of PTPN3 was observed for 53% of samples been reported (15). Our RT-PCR-based gene expression examined. Upon analysis of the clinicopathological information profiling approach permits a more speedy positive for PTPN3-expressing samples, a strong correlation was found identification of target genes in a small tumour specimen. between PTPN3 and the differentiation status of gastric-cancer PTPN3 and PTPN4 are 2 closely-related non-receptor tissues. It thus appears that PTPN3 is expressed more tyrosine phosphatases with a C-terminal tyrosine substantially for the intestinal type of gastric cancer (Lauren’s phosphatase catalytic domain and an N-terminal domain classification) and less so for differentiated cells (p=0.001 and homologous to the band 4.1 superfamily of cytoskeletal p=0.037, respectively, Table I). associated proteins (18). Two additional protein motifs have

1646 Wun et al: PTPN3 and PTPN4 Expression in Human Gastric Cancer

Figure 2. Immunohistochemical staining of PTPN3 tyrosine phosphatase in human gastric cancer sections. Anti-PTPN3 antibody was used for immunohistochemical staining in human gastric cancer tissues by the avidin-biotin-peroxidase complex method, as described in Materials and Methods. Positive PTPN3 tyrosine phosphatase immunohistochemical reactivity was observed in cancer cells as indicated by the reddish-brown color.

also been identified in this family, namely, FERM_C and tumor cells. In terms of the functions of PTPN4, it has been PDZ, the FERM_C domain being the third structural previously reported that PTPN4 function regulation is domain within the FERM domain (28) and structurally located downstream of the glutamate receptor’s signaling similar to the PH and PTB domains (29). The putative location (36). PTPN4 overexpression within COS-7 cells has function of these domains is to provide some sort of linkage previously been reported to be inhibitory to cellular growth, between the and the cytoskeleton (30-32), saturation density and anchorage-independent growth (23), while PDZ may be responsible for specific protein-protein and PTPN4 appears to be a notable tumor suppressor. It interactions (33, 34). PTPN3 is able to interact with valosin- would also be interesting to examine the particular cell type containing protein/p97, a cell-cycle regulator, and thus expressing PTPN4 within human gastric cancer cells and inhibits the growth of NIH3T3 cells (35). The over- their status. The expression ratio between PTPN3 expression of PTPN3 mRNA has previously been shown to and PTPN4 for gastric cancer cell lines might indicate the be correlated with lymph node metastasis for esophageal occurrence of a functional alteration arising during cancer, but inversely correlated with the tumor’s of stomach-derived cancer cells. The precise differentiation status (21). In this report, it was observed molecular function of these 2 phosphatases remains to be that the PTPN3 protein is expressed more substantially for further elucidated. In conclusion, the expression profile intestinal-type gastric cancer. It would be interesting to information obtained will help to identify selected tyrosine examine the mutation status of PTPN3 and the associated phosphatase genes, such as PTPN3 and PTPN4, involved in protein stability, as well as various cellular functions within the development of certain cancers.

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Table I. Relationship between PTPN3 immunostaining and clinico- Acknowledgements pathological features in 92 patients with gastric cancer. This study was supported, in part, by a genome research grant Characteristics (–) (+) p value provided by Academia Sinica, Taiwan (W.-c. Lin) and grants from (n=43; (n=49; the National Science Council (89-2314-B-001-010-M58 and 90- 46%) 53%) 2314-B-001-002-M58), R.O.C.

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1648 Wun et al: PTPN3 and PTPN4 Expression in Human Gastric Cancer

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