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The Nonreceptor-Type Tyrosine Phosphatase PTPN13 Is a Tumor Suppressor Gene in Non–Small Cell Lung Cancer

2012; Elsevier BV; Volume: 180; Issue: 3 Linguagem: Inglês

10.1016/j.ajpath.2011.11.038

1525-2191

Marianna Scrima, Carmela De Marco, Fernanda De Vita, Fernanda Fabiani, Renato Franco, Giuseppe Pirozzi, Gaetano Rocco, Donatella Malanga, Giuseppe Viglietto,

Galectins and Cancer Biology

The aim of the present work was to identify protein tyrosine phosphatases (PTPs) as novel, candidate tumor suppressor genes in lung cancer. Among the 38 PTPs in the human genome that show specificity for phosphotyrosine, we identified six PTPs by quantitative RT-PCR whose mRNA expression levels were significantly down-regulated in lung cancer–derived cell lines (ie, PTPRE, PTPRF, PTPRU, PTPRK, PTPRD, and PTPN13). After validation in primary samples of non–small cell lung cancer (NSCLC), we selected PTPN13 for further studies. The results presented here demonstrate that PTPN13 is a candidate tumor suppressor gene that is frequently inactivated in NSCLC through the loss of either mRNA and protein expression (64/87, 73%) or somatic mutation (approximately 8%). Loss of PTPN13 expression was apparently due to the loss of one or both copies of the PTPN13 locus at 4q (approximately 26% double deletion and approximately 37% single deletion) but not to promoter methylation. Finally, the manipulation of PTPN13 expression in lung cancer cells (ie, NCI-H292, A549) demonstrated that PTPN13 negatively regulates anchorage-dependent and anchorage-independent growth in vitro and restrains tumorigenicity in vivo, possibly through the control of the tyrosine phosphorylation of both EGFR and HER2. In conclusion, the expression screening of PTPs in lung cancer reported here has identified PTPN13 as a novel candidate tumor suppressor in NSCLC whose loss increases signaling from epidermal growth factor receptor and HER2 tyrosine kinase receptors. The aim of the present work was to identify protein tyrosine phosphatases (PTPs) as novel, candidate tumor suppressor genes in lung cancer. Among the 38 PTPs in the human genome that show specificity for phosphotyrosine, we identified six PTPs by quantitative RT-PCR whose mRNA expression levels were significantly down-regulated in lung cancer–derived cell lines (ie, PTPRE, PTPRF, PTPRU, PTPRK, PTPRD, and PTPN13). After validation in primary samples of non–small cell lung cancer (NSCLC), we selected PTPN13 for further studies. The results presented here demonstrate that PTPN13 is a candidate tumor suppressor gene that is frequently inactivated in NSCLC through the loss of either mRNA and protein expression (64/87, 73%) or somatic mutation (approximately 8%). Loss of PTPN13 expression was apparently due to the loss of one or both copies of the PTPN13 locus at 4q (approximately 26% double deletion and approximately 37% single deletion) but not to promoter methylation. Finally, the manipulation of PTPN13 expression in lung cancer cells (ie, NCI-H292, A549) demonstrated that PTPN13 negatively regulates anchorage-dependent and anchorage-independent growth in vitro and restrains tumorigenicity in vivo, possibly through the control of the tyrosine phosphorylation of both EGFR and HER2. In conclusion, the expression screening of PTPs in lung cancer reported here has identified PTPN13 as a novel candidate tumor suppressor in NSCLC whose loss increases signaling from epidermal growth factor receptor and HER2 tyrosine kinase receptors. Lung cancer is the leading cause of cancer deaths worldwide.1Hecht S.S. Tobacco smoke carcinogens and lung cancer.J Natl Cancer Inst. 1999; 91: 1194-1210Crossref PubMed Scopus (1606) Google Scholar, 2Jemal A. Siegel R. Ward E. Hao Y. Xu J. Murray T. Thun M.J. Cancer statistics, 2008.CA Cancer J Clin. 2008; 58: 71-96Crossref PubMed Scopus (10196) Google Scholar Epithelial lung cancer is classified into two main groups: small cell lung cancer (approximately 15% of all lung cancers) and non–small cell lung cancer (NSCLC) (approximately 85% of all lung cancers).3Hirsh F.R. Corrin B. Colby T.V. Tumours of the lung Clinical features and staging.in: Travis W.D. Brambilla E. Muller-Her-melink E. Harris C.C. WHO Classification, Tumours of the Lung, Pleura, Thymus and Heart. IARC Press, Lyon2004: 16-19Google Scholar NSCLC comprises squamous cell carcinoma (SCC), adenocarcinoma (ADC), and large cell cancer (LCC).3Hirsh F.R. Corrin B. Colby T.V. Tumours of the lung Clinical features and staging.in: Travis W.D. Brambilla E. Muller-Her-melink E. Harris C.C. WHO Classification, Tumours of the Lung, Pleura, Thymus and Heart. IARC Press, Lyon2004: 16-19Google Scholar Despite advances in early detection and standard treatment, NSCLC is often diagnosed at an advanced stage, and patients often have poor prognosis, with a 5-year survival rate of 40, low when IS was between 10 and 40, and absent when IS was <10. The following lung cancer cell lines were purchased from ATCC-LGC Promochem (South West London, England): SCC (BEN-1, NCI-H292, NCI-H226, and CALU-1), ADC (A549, NCI-H596, NCI-H2009, NCI-H23, NCI-H522, and NCI-H1975), and LCC (NCI-H460). Cells were maintained in RPMI 1640 (Gibco-Invitrogen, Carlsbad, CA), supplemented with 10% of fetal bovine serum (FBS) and 100 U/mL of penicillin-streptomycin (Life Technologies Inc.). Normal human bronchial epithelial cells (NHBECs) and human small airway epithelial cells (SAECs) were purchased from Cambrex (Milan, Italy) and grown as suggested by the manufacturer.39Reddel R.R. Ke Y. Gerwin B.I. McMenamin M.G. Lechner J.F. Su R.T. Brash D.E. Park J.B. Rhim J.S. Harris C. Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes.Cancer Res. 1988; 48: 1904-1909PubMed Google Scholar Fluorescence in situ hybridization (FISH) analysis was performed on TMAs. BAC clones were designed according to the Ensembl database. TMA slides were deparaffinized, heated in a pressure cooker with 1 mmol/L EDTA (pH 8.0) for 10 minutes, and incubated with pepsin at 37°C for 30 minutes. The slides were then dehydrated in increasing ethanol concentrations and air-dried. The probes were denatured at 96°C for 5 minutes; a hybridization solution was applied on each slide and incubated at 75°C for 1 minute. After overnight incubation at 37°C in a humid chamber, slides were washed with 0.4× standard saline citrate and 0.3% NP40 for 2 minutes at 75°C, air-dried in darkness, and counterstained with DAPI and then coverslips were applied. The BAC clone covering the PTPN13 gene (RP11-64A1) was labeled with dUTP-Spectrum Orange (Vysis Inc., Downers Grove, IL). Control BAC probes covering the chromosome 4 centromeric region was labeled green (KREATECH, Poseidon FISH probes SE4 KBI-20004G D4Z1). Two different investigators who had no previous knowledge of the genetic, clinical, and immunohistochemistry (IHC) results evaluated FISH analysis (R.F. and M.S.). All FISH was scored on an average of 130 nuclei (range, 60 to 210). For evaluation of copy number of PTPN13, samples containing a signal to centromere ratio of 1.0 were classified as disomy, samples containing a signal to centromere ratio of 1.0 but <2 signal spots in at least 50% of cells were classified as monosomy, and samples containing a signal to centromere ratio of <0.75 in at least 50% of cells were considered to carry a loss. This allowed the classification of patients into three groups: FISH disomic, FISH monosomic, and FISH loss. Total RNA and genomic DNA were prepared as previously described.40Chomczynski P. Sacchi N. Single-step method of RNA from isolated by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63148) Google Scholar, 41Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual 2. Cold Spring Harbor Laboratory Press, New York1992Google Scholar Quantitative RT-PCR and quantitative real-time PCR were performed using the Power SYBR Green PCR Master Mix in an ABI Prism 7300 thermocycler (Applied Biosystems, Foster City, CA) according to the manufacturers. cDNA was synthesized from 1 μg of total RNA using QuantiTect Reverse Transcription (Qiagen, Venlo, The Netherlands). The sequence of the primers used in the screening of receptor PTPs (PTPRs, O, E, F, A, M, U, K, J, G and D) and nonreceptor PTPs (PTPNs 4, 14, 6, 9, 21, 23, 4A1, 18, 3, 4A2, and 1) is available on request. For RT-PCR analysis of PTPN13, the primers used were designed on NM_080685.2: forward: 5′-TGGCTCTCCAGGCTGAGTATG-3′ (nt 2114–2134, exon 14) and reverse: 5′-CGGGCAAATAGTGCTCCATT-3′ (nt 2174–2194, exon 15). For gene dosage analysis, primers were designed to amplify 81-bp spanning exon 14 and intron 14 of the PTPN13 gene and subsequently normalized by β-actin: PTPN13 forward: 5′-TGGCTCTCCAGGCTGAGTATG-3′, PTPN13 reverse: 5′AACACCAAACAAAATGGTCCTG-3′, β-actin forward: 5′-TGCGTGACATTAAGGAGAAG-3′, and β-actin reverse: 5′-GCTCGTAGCTCTTCTCC-3′. Each sample was run three times, and each PCR experiment included two nontemplate control wells. The relative amounts of mRNA or DNA were calculated by the comparative CT method.42Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)).Method Methods. 2001; 25: 402-408Crossref PubMed Scopus (123392) Google Scholar Mutation analysis for PTPN13 using direct sequencing performed using the BigDye v3.03 cycle sequencing kit (Applied Biosystems) in a capillary automatic sequencer (ABI PRISM 3100 Genetic Analyzer; Applied Biosystems). Protocols and primers for quantitative real-time PCR and sequencing PTPN13 (exons 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 44, 45, 46, 47, and 48) are available on request. Bisulfite modification of 1 μg of DNA was performed by using the Epitect Bisulfite Kit (Qiagen) according to the manufacturer protocol. Subsequently, bisulfite-treated genomic DNA was amplified using primers designed to amplify nucleotides −214 to −1 of the PTPN13 promoter region: forward: 5′-TTAAGGTATTGGTTGTTATGGTAAT-3′ and reverse: 5′-CACCACTAAAACTCCACCTC-3′. After amplification, the PCR products were directly sequenced. Western blot analysis was performed by standard methods.41Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual 2. Cold Spring Harbor Laboratory Press, New York1992Google Scholar Whole cell extracts were prepared by homogenizing cells in radioimmunoprecipitation assay buffer (9.1 mmol/L dibasic sodium phosphate, 1.7 mmol/L monobasic sodium phosphate, 150 mmol/L NaCl, pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 100 μg/mL of phenylmethylsulfonyl fluoride, and 1 mmol/L sodium orthovanadate) containing protease inhibitors. Lysates were cleared by centrifugation, and proteins were separated by SDS-PAGE. Antibodies used were from Cell Signaling Technology [anti-pEGFR (no. 3777), anti-EGFR (no. 2232), anti-pHER2 (Y1228, no. 2247), anti-HER2 (no. 2242), anti-pMAPK (no. 4370), anti-MAPK (no. 4695), anti-AKT1 (no. 2938), and anti-pAkt (S473, no. 9277)], Santa Cruz Biotechnology (Santa Cruz, CA) [anti-PTPN13 (H300)], or Novus Biologicals (Littleton, CO) [anti-pHER2 (Y877, no. NB100-92549)]. Mouse and rabbit monoclonal special secondary antibodies were from Santa Cruz Biotechnology. The human PTPN13 (NM_006264) MISSION short hairpin RNA (shRNA) set (Sigma-Aldrich, St. Louis, MO) and the mission nontarget control transduction virus (SHC002V; Sigma-Aldrich) were used to generate lentiviral particles in HEK293T packaging cells. Subconfluent HEK293T cells were co-transfected with 13 μg of PTPN13 MISSION shRNA set, 18 μg of pCMV-deltaR8.91, and 12 μg of pCMV-VSVG.36 After transfection, supernatants were collected at 8-hour intervals, filtered, and used for three rounds of transduction of NCI-H292 cells in the presence of 8 μg/mL of polybrene (Sigma-Aldrich). Transduced cells underwent three rounds of infection (8 hours each round) and were selected in medium containing 1 μg/mL of puromycin. The PTPN13 expression plasmid was a generous gift from Dr. Carl-Henrik Heldin (Ludwig Institute for Cancer Research, Uppsala University, Uppsala, Sweden). The pcDNA3-HER2 and pcDNA3-EGFR plasmids were purchased at Addgene. A549 cells were transfected with Trans-LT1 transfection reagent (Mirus, Madison, WI), according to the manufacturer's protocol. In most experiments transfection efficiency was approximately 50% to 60%, as demonstrated by parallel transfection with plasmid-enhanced green fluorescent protein (Clontech Laboratories, Mountain View, CA). Cell proliferation was assayed by MTT (Sigma-Aldrich) reduction. Cells were plated in 96-well, flat-bottomed microtiter plates (200-μL cell suspensions, 2 × 103 per well for NCI-H292) and incubated with MTT substrate (5 mg/mL) for 4 hours. Every 24 hours, the culture medium was removed and anhydrous 2-propanol was added. Optical density was measured at 570 nm. NCI-H292 cells (5 × 103) were resuspended in RPMI plus 10% FBS, containing 0.35% low-melting agarose (type VII; Sigma-Aldrich) and seeded onto 0.5% low-melting agarose in Dulbecco's modified Eagle's medium containing 10% FBS. Colonies were scored 3 weeks after preparation and stained with 0.1% crystal violet (Sigma-Aldrich). Quantification (mean and SD) was from three independent experiments. NCI-H292 cells (1 × 106) were resuspended in 100 μL of 10% FBS and 100 μL of Matrigel (BD Biosciences, Franklin Lakes, NJ) and subcutaneously injected into the right flank of 6-week-old, athymic, nude mice (Charles River, Wilmington, MA) in triplicate. Every 7 days tumor size was measured with a caliper. Cell were transfected with pBABE (1 μg) in the presence of PTPN13 or control empty vector (4 μg) by Trans-LT1 transfection reagent (Mirus), according to the manufacturer. After 48 hours, cells were selected with 1 μg/mL of puromycin (Sigma) for approximately 20 days, stained with crystal violet solution for 10 minutes, and counted. The aim of the study was to identify tyrosine phosphatases that contribute to the development of NSCLC. A recent survey found that 107 PTPs are encoded by the human genome.25Alonso A. Sasin J. Bottini N. Friedberg I. Friedberg I. Osterman A. Godzik A. Hunter T. Dixon J. Mustelin T. Protein tyrosine phosphatases in the human genome.Cell. 2004; 117: 699-711Abstract Full Text Full Text PDF PubMed Scopus (1519) Google Scholar Of these, 38 belong to the subgroup of "classic PTPs" that show specificity for phosphotyrosine. By analysis of the Gene Expression Atlas (ArrayExpress Atlas), a meta-analysis–based database of the ArrayExpress Archive, we selected 22 PTPs that are expressed in normal lung among the subgroup of classic PTPs: 10 receptor PTPs (PTPRs, O, E, F, A, M, U, K, J, G and D) and 12 nonreceptor PTPs (PTPNs 13, 4, 14, 6, 9, 21, 23, 4A1, 18, 3, 4A2, and 1). Subsequently, we performed a preliminary quantitative RT-PCR screening of the mRNA expression of the 22 selected PTPs in ADC- or SCC-derived cell lines compared with NHBECs (Figure 1). The cell lines used were as follows: ADC-derived cell lines, A549, NCI-H522, NCI-H2009, NCI-H23, NCI-H460, and NCI-H596; and SCC-derived cell lines, NCI-H292, NCI-H226, CALU1, and BEN1. The quantitative RT-PCR analysis of NSCLC-derived cell lines identified six different PTPs with significant down-regulated expression: five receptor PTPs [PTPRE (P = 0.01), PTPRF (P = 0.001), PTPRU (P = 0.02), PTPRK (P = 0.0005), and