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Expression Profile of Tyrosine Phosphatases in HER2 Breast

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Expression Profile of Tyrosine Phosphatases in HER2 Breast Cellular Oncology 32 (2010) 361–372 361 DOI 10.3233/CLO-2010-0520 IOS Press Expression profile of tyrosine phosphatases in HER2 breast cancer cells and tumors Maria Antonietta Lucci a, Rosaria Orlandi b, Tiziana Triulzi b, Elda Tagliabue b, Andrea Balsari c and Emma Villa-Moruzzi a,∗ a Department of Experimental Pathology, University of Pisa, Pisa, Italy b Molecular Biology Unit, Department of Experimental Oncology, Istituto Nazionale Tumori, Milan, Italy c Department of Human Morphology and Biomedical Sciences, University of Milan, Milan, Italy Abstract. Background: HER2-overexpression promotes malignancy by modulating signalling molecules, which include PTPs/DSPs (protein tyrosine and dual-specificity phosphatases). Our aim was to identify PTPs/DSPs displaying HER2-associated expression alterations. Methods: HER2 activity was modulated in MDA-MB-453 cells and PTPs/DSPs expression was analysed with a DNA oligoar- ray, by RT-PCR and immunoblotting. Two public breast tumor datasets were analysed to identify PTPs/DSPs differentially ex- pressed in HER2-positive tumors. Results: In cells (1) HER2-inhibition up-regulated 4 PTPs (PTPRA, PTPRK, PTPN11, PTPN18) and 11 DSPs (7 MKPs [MAP Kinase Phosphatases], 2 PTP4, 2 MTMRs [Myotubularin related phosphatases]) and down-regulated 7 DSPs (2 MKPs, 2 MTMRs, CDKN3, PTEN, CDC25C); (2) HER2-activation with EGF affected 10 DSPs (5 MKPs, 2 MTMRs, PTP4A1, CDKN3, CDC25B) and PTPN13; 8 DSPs were found in both groups. Furthermore, 7 PTPs/DSPs displayed also altered protein level. Analysis of 2 breast cancer datasets identified 6 differentially expressed DSPs: DUSP6, strongly up-regulated in both datasets; DUSP10 and CDC25B, up-regulated; PTP4A2, CDC14A and MTMR11 down-regulated in one dataset. Conclusions: Several DSPs, mainly MKPs and, unexpectedly, MTMRs, were altered following HER2-modulation in cells and 3 DSPs (DUSP6, CDC25B and MTMR11) were altered in both cells and tumors. Among these, DUSP6, strongly up-regulated in HER2-positive tumors, would deserve further investigation as tumor marker or potential therapy target. Keywords: Tyrosine phosphatases, phosphorylation, HER2, breast cancer, array analysis 1. Introduction motility and invasion may be up-regulated [30,39,42]. However, the way HER2 over-expression deregulates About 10% of human breast cancers display over- these multiple signaling pathways is still poorly un- expressed HER2 (human epidermal growth factor re- derstood [19,39]. Within this background, PTPs (pro- ceptor, ERBB2), a member of the ERBB receptor tein tyrosine phosphatases) and DSPs (dual-specificity family [13,17]. Such tumors are highly invasive and phosphatases) might play a role, since members of metastatic, and generally do not respond to anti- these families are signaling molecules in RTK (recep- estrogen treatments [28,29]. Studies in tumors and tor tyrosine kinase) pathways. tumor-derived cell lines indicated that HER2 overex- The human genome sequence revealed that only 38 pression is mainly due to gene amplification. HER2 PTPs exist, namely 21 receptor and 17 soluble PTPs. repression, using tyrosine kinase inhibitors or mono- DSPs are approximately 65 [32] and include members clonal antibodies, is a valuable therapeutic approach of the MKPs, SSH, PTP4A, CDC14, PTEN, Myotubu- [3,20]. HER2 activation evokes stimulation of both larins, CDC25 and EYA families [1,2]. PTPs and DSPs MAPK and PI3K/AKT pathways, leading to prolifer- are positive or negative regulators in a wide range of ation and survival [40]. Also pathways governing cell signaling networks, thus contributing to control cell * proliferation, adhesion and migration, differentiation Corresponding author: Emma Villa-Moruzzi, PhD, Depart- and survival [15,32]. ment of Experimental Pathology, via Roma 55, 56126 Pisa, Italy. Tel.: +39 050 2218 551; Fax: +39 050 2218 557; E-mail: In cancer pathways, PTPs and DSPs generally play [email protected]. an anti-oncogenic role, by terminating the action of 1570-5870/10/$27.50 © 2010 – IOS Press and the authors. All rights reserved 362 M.A. Lucci et al. / Tyrosine phosphatases in HER2 breast cancer cells and tumors PTKs (protein tyrosine kinases). Examples are the Placid, NY, USA) and CDKN3 (BD Biosciences, MKPs (i.e., DSPs that dephosphorylate MAPKs) [14] Franklin, NJ, USA). The anti-CDC25C antibodies and several PTPs [32]. However, PTPs may also trans- were described previously [35]. The anti-PTPRA rab- duce positive signals, thus favoring oncogenesis. Ex- bit polyclonal antibodies were a kind gift from Dr. amples are PTPN11 [23], PTPN1 [33], PTPRE [11] J. den Hertog (Utrecht, The Netherlands). and the cell cycle regulators CDC25C, B and A [6]. In any event, PTPs and DSPs must be regulated to pre- vent havoc in the cell. Little is still known on PTPs 2.2. Cell culture, treatments and extracts and DSPs in breast cancer, particularly in the HER2- dependent type. Several studies focused on PTEN, The MDA-MB-453 cells were grown in RPMI sup- a tumor suppressor which is often mutated in tumors plemented with 10% fetal calf serum [36]. The MDA- [25]. A role in favoring HER2-evoked tumorigenesis MB-453 cells carrying the intracellular expression of was also reported for PTPN11 [21,43] and PTPN1 the p185HER2-specific single chain antibody (5R) and [4]. Other studies indicated a role for PTPN18 [10] in control MDA-MB-453 cells carrying the empty vec- HER2-overexpressing tumor cell lines and for PTPRE tor (LBSN) were grown in DMEM supplemented with in neu mice [11]. However, many more PTPs and DSPs 10% fetal calf serum and 0.5 mg/ml geneticin [8]. might be involved, given the complexity of the ERBB In brief, cDNA encoding the p185HER2-specific single receptors signaling. chain antibody 5R was cloned into the LXSN retroviral PTPs and DSPs alterations in cancer may com- vector and used to infect the packaging cell line gp + prise changes in the expression and protein levels, AM12. Following G418 selection the virus-containing or mutations, as described in the case of colon can- medium was collected and used to infect the MDA- cer [38]. Post-translation modifications may also oc- MB-453 cell line. The infection cycle was repeated cur. The present project was undertaken to iden- three times and cells were tested for p185HER2 surface tify PTPs and DSPs displaying altered expression expression by FACScan analysis. For EGF-treatment, levels in HER2-overexpressing human breast can- sub-confluent cells were serum-starved and exposed cer and cancer-derived cell lines. We analyzed the to 50 ng/ml EGF (diluted in DMSO) or to DMSO PTPs and DSPs in: (1) MDA-MB-453 cells (HER2- alone (time 0). For immunoblotting, the cells were overexpressing human breast cancer cells) carrying washes in cold PBS and lysed in 50 mM TRIS-HCl, pH permanent HER2 inhibition or activated with EGF 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and (2) 2 public datasets of primary invasive HER2- positive breast tumors. The aim was to identify PTPs 7.5 mM 2-mercaptoethanol, added with 1 mM ortho- and DSPs displaying HER2-associated alterations, that vanadate, 50 mM NaF and protease inhibitors (0.02% might sustain tumor biology features, such as survival benzamidine, 0.02% PMSF, 0.02% TPCK, 10 µg/ml and motility, and might represent new tumor markers soybean trypsin inhibitor and 4 µg/ml leupeptine). Cell or potential therapy targets. extracts were then diluted 1:2 in Laemmli buffer and boiled. 2. Materials and methods 2.3. RNA extraction and cDNA/cRNA preparation 2.1. Materials RNA was purified from cells grown on 30 mm plates Tissue culture media and additives, fetal calf serum, using the SV total RNA isolation system (Promega, EGF, the antibodies to α-tubulin, peroxidase-conjugat- Madison, WI, USA). Labeled antisense RNA (cRNA) ed antibodies and protein A-peroxidase were pur- was synthesized using the TrueLabeling-AMP 2.0 kit chased from Sigma Aldrich (Munich, Germany). The (SABiosciences, formerly SuperArray Biotech, Fred- other commercial antibodies were as follows: phospho- erick, MA, USA). In brief, cDNA was prepared by Y1248-HER2, pan-EGFR, DUSP1, DUSP2, DUSP6, reverse transcription of total RNA (2–3 µg) and imme- MTM1 and PTEN (Santa Cruz Biotech, Heidelberg, diately used for cRNA synthesis, labeling and ampli- Germany); pan-HER2 (Cell Signaling Technology, fication in the same tube. All the rest was according Danvers, MA, USA); phospshoY1173-EGFR (Nan- to the kit instructions. Four h incubation yielded be- otools, Teningen, Germany); PTPN11 (Upstate, Lake tween 4 and 12 µg of cRNA. M.A. Lucci et al. / Tyrosine phosphatases in HER2 breast cancer cells and tumors 363 2.4. DNA arrays branes were previously treated with 5 mM phosphate buffer, 2% SDS and 2 mM 2-mercaptoethanol at 60◦C Custom oligo GEArray, displaying 60-mer oligonu- for 15 min. For immunoblot quantification, the PTP cleotide probes, targeting 103 PTPs and DSPs and or DSP protein bands from treated and control cells 5 housekeeping genes (RPS27A, GAPDH, B2M, were analyzed by densitometric scanning and normal- HSP90AB1 and ACTB), were produced by SABio- ized to the α-tubulin band analyzed in the same ex- sciences. cRNA (2 µg) was used for overnight hy- periment (i.e., the PTP or DSP/α-tubulin value ratios bridization, followed by peroxidase-based chemilumi- were calculated). Normalized data from different ex- nescence detection on X-ray films, according to the periments were averaged, as specifically indicated. Re- manufacturer’s instructions. Data extraction, normal- sults are given as percent of averages from control cells ization with the indicated housekeeping genes and (controls were LBSN cells in Fig. 1 and time 0 cells in analysis used the GEArray Expression Analysis Suite Fig. 3). (SABiosciences). Expression levels are given as the ra- tio of treated to control cells and we reported changes greater than 30%, a boundary selected to boost the re- 2.7. Public gene expression data analysis sults reliability.
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