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Original Article Knockdown of Protein Tyrosine Phosphatase Receptor U Inhibits Growth and Motility of Gastric Cancer Cells

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Original Article Knockdown of Protein Tyrosine Phosphatase Receptor U Inhibits Growth and Motility of Gastric Cancer Cells Int J Clin Exp Pathol 2014;7(9):5750-5761 www.ijcep.com /ISSN:1936-2625/IJCEP0001557 Original Article Knockdown of protein tyrosine phosphatase receptor U inhibits growth and motility of gastric cancer cells Yongjie Liu1, Zhichuan Zhu1, Zhiqi Xiong1, Jing Zheng1, Zelan Hu1, Jiangfeng Qiu2 1Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Tech- nology, Shanghai 200237, P. R. China; 2Department of Gastrointestinal Surgery, Ningbo First Hospital, Ningbo 315010, P. R. China Received July 26, 2014; Accepted August 23, 2014; Epub August 15, 2014; Published September 1, 2014 Abstract: Protein tyrosine phosphatase receptor U (PTPRU) has been shown to be a tumor suppressor in colon cancer by dephosphorylating β-catenin and reducing the activation of β-catenin signaling. Here, we investigate the expression of PTPRU protein in gastric cancer cell lines, gastric cancer tissues and respective adjacent non-cancer tissues and find that the 130kDa nuclear-localized PTPRU fragment is the main PTPRU isoform in gastric cancer cells, whereas the full-length PTPRU is relatively lowly expressed. The level of the 130kDa PTPRU is higher in gastric cancer tissues than in adjacent non-cancer tissues. Knockdown of endogenous PTPRU in gastric cancer cells us- ing lentivirus-delivered specific shRNA results in the attenuation of cell growth, migration, invasion and adhesion. Knockdown of PTPRU also inhibits tyrosine phosphorylation and transcriptional activity of β-catenin as well as levels of focal adhesion proteins and lysine methylation of histone H3. These results indicate that PTPRU is required for gastric cancer progression and may serve as a potential therapeutic target. Keywords: PTPRU, gastric cancer, proliferation, motility, β-catenin Introduction adhesion, apoptosis, migration, and invasion [3-5]. The role of PTKs in cancers is well charac- Gastric cancer is the second most common terized and their inhibitors have been utilized as cancer type and one of the deadliest epithelial anticancer drugs [6]. Although the role of PTPs malignancies, with a 5-year survival rate of in cancers is not as well studied as that of PTKs, approximate 24% worldwide and high frequen- growing evidence suggests that functional cy of occurrence in Eastern Asia [1, 2]. Currently, aberration of PTPs is associated with the pro- surgical resection remains the most important gression of multiple types of human cancers way to prolong the survival time of gastric can- [7-12]. The PTP superfamily can be divided into cer patients, but nevertheless with a relapse several subfamilies based on the structural rate of more than 50%, especially in patients diversity. Among them, the R2B RPTP subfamily with lymph node metastasis [2]. Compared comprises four members, PTPRU, PTPRM, with other more intensively investigated can- PTPRT and PTPRK. A myriad of reports indicate cers, such as breast cancer, colorectal cancer that R2B RPTPs are negative regulators in and glioma, the molecular mechanisms under- tumorigenesis. For example, PTPRK is mutant lying gastric cancer progression are largely in sporadic endocrine pancreatic tumors, glio- unclear so far. Therefore, developing new strat- ma, and primary central nervous system lym- egies to complement the shortcoming of the phomas [13-15]. It also negatively regulates the current therapies is warranted. proliferation and motility of melanoma and breast cancer cells [12, 16]. The aberrant func- Tyrosine phosphorylation as a molecular switch, tion of PTPRM is associated with the progres- is dynamically regulated by protein tyrosine sion of ovarian carcinoma, prostate cancer, phosphatase (PTPs) and kinases (PTKs). It breast cancer and glioma [17-20]. PTPRT is the plays an important role in various cellular sig- most frequently mutated phosphatase gene in nalings that affect growth, differentiation, cancers including colon, lung, skin and stomach Tyrosine phosphatase receptor U and gastric cancer [21, 22]. Similar to other members of the R2B and 0.25 mg/ml sodium deoxycholate) supple- RPTP subfamily, PTPRU negatively regulates mented with PMSF (Beyotime, Nantong, China), the growth and migration of colon cancer cell phosphatase inhibitor (Sangon, Shanghai, SW480 [23, 24]. In contrast to the tumor sup- China) and protease inhibitor cocktail (Thermo pressive role of full-length and mutant R2B Scientific, Waltham, USA), then mixed with load- RPTPs, some of their proteolytic fragments ing buffer and boiled. Nuclear and cytoplasmic contribute to tumorigenesis [20, 25, 26]. protein fractions were prepared using the Intracellular fragments of PTPRK and PTPRM nuclear and cytoplasmic protein extraction kit generated by furin-initiated proteolytic cleav- (Beyotime). Equal amount of proteins were sep- age can translocate to the nucleus and activate arated by SDS-PAGE and transferred onto poly- the oncogenic signaling [25, 26]. PTPRM frag- vinylidenedifluoride membranes (Millipore, ments positively regulate the growth and motil- Billerica, USA). Membranes were blocked in 5% ity of glioma cells probably in this kind of way nonfat dry milk or bovine serum albumin in [20, 26]. Together, these findings suggest a TBST (Tris-buffer saline with 0.05% Tween-20) pro-oncogenic role of the proteolytic fragments for 1 h, then incubated overnight at 4°C with of R2B RPTPs. antibodies against PTPRU (Sigma-Aldrich, St. Louis, USA), PTPλ (Abcam, Cambridge, UK), The expression, function and mechanism of β-catenin, EGFR, FAK (BD Biosciences, San PTPRU in cancers are not as well characterized Jose, USA), c-Myc, Dnmt1, p-ERK1/2 (T202/ as other R2B RPTPs. In the present study, we Y204), H3K27me2, Histone H3, p-Tyr-102, TCF4 found that the expression of a 130KDa PTPRU (Cell Signaling Technology, Beverly, USA), isoform is higher in gastric cancer tissues than ERK1/2, H3K9me3, paxillin, PYK2 (Bioworld respective adjacent non-cancer tissue. Knoc- Technology, Nanjing, China), cyclin B1, cyclin kdown of PTPRU suppresses growth and motil- D1, VE-cadherin (Boster, Wuhan, China) and ity and β-catenin transcriptional activity of gas- GAPDH (Kangchen, Shanghai, China). Corr- tric cancer cells. We conclude that endogenous esponding horseradish peroxidase-conjugated PTPRU play oncogenic roles in gastric cancer anti-mouse and anti-rabbit secondary antibod- and may be of clinical value. ies (Cell Signaling Technology, Beverly, USA) Materials and methods were used. Protein bands were visualized by photo development using Kodak film. To ana- Tissue samples lyze the relative levels of PTPRU, band gray- scales of PTPRU and GAPDH were quantified A total of 24 pairs of gastric cancer (GC) and using the Adobe Photoshop software and the their adjacent non-cancer tissues were collect- ratio of PTPRU to GAPDH was calculated. ed immediately after surgery from Department of Gastrointestinal Surgery, Ningbo First Lentivirus-mediated shRNA knockdown Hospital after surgery and stored at -80°C until being used. This work was approved by the Lentivirus encoding shRNA plasmids (on vector Ethics Committee of Ningbo First Hospital. pLKD-CMV-GFP-U6-shRNA, Neuron Biotech, Shanghai, China) specifically targeting human Cell culture PTPRU and a scrambled shRNA plasmid were The human gastric cancer cell lines AGS, packed by Neuron Biotech. Western blot and SGC7901, MKN45 and MGC803 were cultured real time PCR were used to validate the knock- in RPMI 1640 medium (Corning, Williamsburg, down efficacy and found that the most efficient USA) supplemented with 10% fetal bovine shRNA sequence was: CCGGGGAGATGATC- serum (Life technologies, Grand Island, USA), CGCATTGATCCCTCAGGGATCAATGCGGATCATCT- 100U/ml penicillin, 0.1 mg/ml streptomycin at CCTTTTTTG. The sequence of the scrabled shRNA was: CCGGTTCTCCGAACGTGTCACG- 37°C in a 5% CO2 incubator. These cell lines were obtained from the cell bank of the Chinese TTTCAAGAGAACGTGACACGTTCGGAGAATTT- Academy of Sciences (Shanghai, China). TTTG. Western blot Immunofluorescence Cells and tissues were collected and lysed on Cell suspension was plated onto sterile glass ice in lysis buffer (20 mM Tris-HCl pH 7.5, 2 mM coverslips in 6-well plates for 24 h, then fixed EDTA, 1% NP-40, 150 mM NaCl, 1 mg/ml SDS with 4% paraformaldehyde at 4°C for 15 min. 5751 Int J Clin Exp Pathol 2014;7(9):5750-5761 Tyrosine phosphatase receptor U and gastric cancer Cells were incubated in turn with blocking buf- were harvested, fixed with 70% (v/v) ethanol fer (3% bovine serum albumin and 0.1% Triton overnight at -20°C, incubated with 50 µg/ml in phosphate-buffered saline) for 1 h, with RNase A (Thermo Scientific) and 50 µg/ml PTPRU or PTPλ antibody at 4°C overnight, with propidium iodide (Sigma-Aldrich) in PBS for 30 biotin labeled secondary antibody (Life tech- min at 37°C in the dark and filtered by sieves to nologies) for 2 h, and with Cy3 labeled avidin get single-cell suspension. Stained nuclei were (Life technologies) and Hoechst 33342 analyzed by a FACSCalibur Flow Cytometry with (Beyotime) for 2 h. Fluorescent images were 10,000 gated events/determination. DNA ploi- taken using a Nikon eclipse Ti-S inverted dy was analyzed by FlowJo software. microscope. Wound-healing assay Real-time quantitative PCR Cells were seeded onto a 6-well plate and Total RNA was extracted using Trizol (Life tech- scraped with a 10 µl pipet tip when they grew nologies), and reverse transcribed using RT into confluent cell monolayer. Culture medium M-MLV (Promega, Beijing, China) with oligo dT. was replaced with serum free RPMI 1640 to Quantitative real-time PCR with SYBR
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