Oncogene (2014), 1–13 © 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

ORIGINAL ARTICLE

Protein tyrosine phosphatase PTPN3 inhibits lung cancer cell proliferation and migration by promoting EGFR endocytic degradation M-Y Li1,2, P-L Lai1, Y-T Chou3, A-P Chi1, Y-Z Mi1, K-H Khoo1,2, G-D Chang2, C-W Wu3, T-C Meng1,2 and G-C Chen1,2 Epidermal growth factor receptor (EGFR) regulates multiple signaling cascades essential for cell proliferation, growth and differentiation. Using a genetic approach, we found that Drosophila FERM and PDZ domain-containing protein tyrosine phosphatase, dPtpmeg, negatively regulates border cell migration and inhibits the EGFR/Ras/mitogen-activated protein kinase signaling pathway during wing morphogenesis. We further identified EGFR pathway substrate 15 (Eps15) as a target of dPtpmeg and its human homolog PTPN3. Eps15 is a scaffolding adaptor protein known to be involved in EGFR endocytosis and trafficking. Interestingly, PTPN3-mediated tyrosine dephosphorylation of Eps15 promotes EGFR for lipid raft-mediated endocytosis and lysosomal degradation. PTPN3 and the Eps15 tyrosine phosphorylation-deficient mutant suppress non-small-cell lung cancer cell growth and migration in vitro and reduce lung tumor xenograft growth in vivo. Moreover, depletion of PTPN3 impairs the degradation of EGFR and enhances proliferation and tumorigenicity of lung cancer cells. Taken together, these results indicate that PTPN3 may act as a tumor suppressor in lung cancer through its modulation of EGFR signaling. Oncogene advance online publication, 29 September 2014; doi:10.1038/onc.2014.312

INTRODUCTION Reversible tyrosine protein phosphorylation by protein tyrosine kinases and protein tyrosine phosphatases (PTPs) acts as a molecular switch that regulates a variety of biological processes.1,2 The receptor tyrosine kinase epidermal growth factor receptor (EGFR), the best characterized member of the ErbB family receptors, acts as a critical regulator of numerous cellular processes, including growth, proliferation and differentiation. Upon activation by its growth factor ligands, EGFR undergoes dimerization and activation, leading to tyrosine phosphorylation of the intracellular region of the receptor as well as many cytoplasmic substrates.3,4 The activated EGFR is then internalized by clathrin-mediated endocytosis and sorted into the endosomal compartments, through which it is either recycled back to the plasma membrane or transported to the lysosome for degradation.5 Because overexpression or constitutive activation of EGFR has been implicated in the pathogenesis and progression of a variety of human malignancies,6 it is therefore crucial to understand how EGFR signaling is regulated. Several PTPs have been implicated in the regulation of EGFR signaling. Among them, PTPRK, DEP-1 (PTPRJ), PTP1B (PTPN1), SHP-1 (PTPN6), TCPTP (PTPN2), PTPN9 and PTPN12 have been shown to downregulate EGFR signaling by dephosphorylating EGFR.7–11 The receptor-type PTP DEP-1 dephosphorylates EGFR on the cell surface and inhibits its internalization.7 On the other hand, the endoplasmic reticulum-localized PTP1B has been reported to regulate EGFR signaling from endosomes.12,13 PTP1B promotes the sequestration of EGFR onto internal vesicles of multivesicular bodies.14 The ESCRT (endosomal sorting complex required for transport) complexes are known to play an important role in 1

sorting EGFR to multivesicular bodies.15 Recently, Ali et al.16 showed that the ESCRT accessory protein HD-PTP/PTPN23 coordinates with the ubiquitin-specific peptidase UBPY to drive EGFR sorting to the multivesicular bodies. A better understanding of the role of PTPs in regulating EGFR signaling will help to provide insights into the molecular mechanisms behind EGFRmediated tumorigenesis. PTPN3 (PTPH1) and the closely-related PTPN4 (PTPMEG) are non-transmembrane PTPs that contain an N-terminal FERM (Band 4.1, Ezrin, Radixin, Moesin homology) domain followed by a single PDZ (PSD95, Dlg, ZO1) domain and the C-terminal PTP domain.1 They have been implicated in the regulation of cell growth and proliferation.17,18 However, their role in receptor protein tyrosine kinase signaling is not clear. The dPtpmeg is the Drosophila homolog of mammalian PTPN3 and PTPN4. Phenotypic analyses have revealed that dptpmeg mutants exhibit aberrant mushroom body axon projection patterns in the brain.19 Besides its role in regulating neuronal wiring, the molecular function of dPtpmeg has remained largely unknown. In this study, we identified EGFR pathway substrate 15 (Eps15) as a substrate of dPtpmeg and PTPN3. Eps15 is known to be an endocytic adaptor involved in the regulation of EGFR trafficking.20 PTPN3 dephosphorylated Eps15 and promoted EGFR for lipid raft-mediated endocytosis and lysosomal degradation. The ectopic expression of PTPN3 or Eps15-Y850F mutant in the non-small-cell lung cancer (NSCLC) cells inhibited cell proliferation, migration and tumor growth. Our findings uncover a novel role for PTPN3 in the regulation of EGFR endocytic trafficking, degradation and signaling.

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; 2Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan and Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan. Correspondence: Dr G-C Chen, Institute of Biological Chemistry, Academia Sinica, 128 Academia Road, Section 2, Taipei 115, Taiwan. E-mail: [email protected] Received 3 April 2014; revised 26 July 2014; accepted 16 August 2014 3

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RESULTS Drosophila Ptpmeg is involved in regulating the EGFR signaling pathway We previously performed genetic analyses to identify nontransmembrane PTPs that could modulate border cell migration during Drosophila oogenesis.21 Although RNA interference (RNAi)mediated downregulation of Drosophila non-transmembrane PTPs did not have an obvious effect on border cell migration at stage 10 egg chambers,21 we found that dptpmeg mutation caused accelerated migration of border cells and the clusters reached oocyte prematurely at stage 9 (Figures 1a and b). The receptor tyrosine kinases, platelet-derived growth factor (PDGF)- and vascular endothelial growth factor (VEGF)-related receptor (PVR) and EGFR, have been shown to play an important role in guiding migration of the border cells toward the oocyte.22,23 As shown in Figures 1c and d, ectopic expression of dPtpmeg with the border cell-specific Slbo-Gal4 driver impaired border cell motility. Moreover, clonal analysis revealed elevated phosphotyrosine levels in dptpmeg homozygous mutant border cell clones (marked by the loss of green fluorescent protein (GFP); Figure 1e, e’), suggesting that dPtpmeg may antagonize receptor tyrosine kinase-mediated tyrosine phosphorylation and border cell migration. It has been reported that EGFR signaling regulates a variety of developmental processes in Drosophila, including wing vein formation.24,25 To investigate the role of dPtpmeg in vein patterning, we used engrailed-Gal4 (en-Gal4) driver to restrict dPtpmeg expression in the posterior compartment of the wing; therefore, the anterior part served as control. Interestingly, expression of wild-type dPtpmeg, but not phosphatase-deficient mutant (dPtpmeg-CS), caused a wing vein missing phenotype (Figures 1f–h). The dPtpmeg-induced wing vein defects could be rescued by coexpressing dPtpmeg-RNAi (Figure 1i). Moreover, ectopic expression of PTP61F,26 the Drosophila homolog of PTP1B, in the developing wing did not affect the normal pattern of veins (Figure 1j). These results together suggest that the wing vein defects are specifically caused by dPtpmeg in a phosphatase activity-dependent manner. To determine the relationship between dPtpmeg and EGFR signaling, we examined whether the dPtpmeg-induced wing vein defects could be modulated by components of the EGFR/Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Genetic analysis revealed that the wing vein missing phenotype caused by dPtpmeg misexpression can be suppressed by the gain-of-function mutation EgfrElp, by the reduction of Ras inhibitor RasGAP1 or by the coexpression of

the activated form of Drosophila Ras (RasV12) (Figures 1k–m), but not by coexpressing the constitutively active phosphoinositide 3kinase (Dp110-CAAX) or active Akt (myr-Akt) (Figures 1n–o). EGFR is known to induce extracellular signal-regulated kinase/MAPK phosphorylation in future vein regions during wing development.27 Strikingly, we found that clonal expression of dPtpmeg in wing imaginal discs using the flipout/Gal4 system28 led to a marked reduction of MAPK phosphorylation in GFP-positive dPtpmeg-expressing cells (Figures 1p, p’). Taken together, these data indicate that dPtpmeg acts as a negative regulator of the EGFR/Ras/MAPK signal pathway. Identification of Eps15 as a substrate of dPtpmeg We have recently established a mass spectrometry (MS)-based substrate trapping strategy to identify putative substrates of PTP61F, the smallest non-transmembrane PTP in Drosophila.26 We used the same approach to identify potential substrates of dPtpmeg. The bacterially expressed wild-type (WT) form and the substrate-trapping DA mutant form of dPtpmeg were incubated with Drosophila S2 cell lysates. After immunoprecipitation of dPtpmeg, the associated proteins were eluted, resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and subjected to liquid chromatography tandem mass spectrometry analysis. Several proteins, including Eps15, Pvr, Cortactin and Ter94 (mammalian VCP/p97), were identified to interact specifically with the dPtpmeg-DA mutant (Figure 2a). Interestingly, previous work has identified VCP/p97 as a substrate of PTPN3 in mammalian cells.17 Moreover, the identification of Pvr as a putative substrate of dPtpmeg is consistent with the finding that dPtpmeg plays a role in regulating border cell migration (Figures 1a–d). Here we have focused our study on Eps15. Eps15 is a multidomain adaptor protein that contains EH domains at the N-terminus and two ubiquitin-interacting motifs at the C-terminus.20 Eps15 associates with components of the AP1 and AP2 complexes and plays an important role in clathrin-mediated endocytosis.20 To understand the relationships between dPtpmeg and Eps15, we examined whether dPtpmeg genetically interacted with Eps15. Ectopic expression of Eps15 in the developing wing with en-Gal4 caused a wing-notching phenotype. Notably, the Eps15-induced wing defects could be suppressed by coexpression of dPtpmeg (Supplementary Figure S1A), suggesting that dPtpmeg has an antagonist effect on Eps15. The activation of EGFR has been

Figure 1. Drosophila Ptpmeg negatively regulates EGFR signaling. (a) Fascin staining in a control and dPtpmeg mutant egg chamber at stage 9. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue) and actin was stained with phalloidin (red). Bar, 50 μm. (b) Quantification of the percentage of stage 9 border cell clusters that prematurely reach the oocyte in the indicated genotypes. Each stage 9 egg chamber was divided into three regions: 100% motility, 450% motility and o50% motility. Border cells of stage 9 egg chambers were scored and the percentage was presented in histogram form (N4100 for each genotype). (c) Control (Slbo-Gal44UAS-mCD8-GFP) and dPtpmeg (SlboGal44UAS-mCD8-GFP/UAS-dPtpmeg) stage 10 egg chambers were stained with DAPI (blue) and phalloidin (red). (d) Quantification of border cell migration of the indicated genotypes in (c). To define defects in border cell migration, each stage 10 egg chamber was divided into three regions: 100% motility, 450% motility and o50% motility. Border cells of stage 10 egg chambers were scored and the percentage was presented in histogram form (N4100 for each genotype). BC, border cell. (e) A marked increase in phospho-Tyrosine (pTyr) levels (arrowhead) in dPtpmeg homozygous mutant border cell clones (GFP-negative cells). The dPtpmeg mutant clone was marked with white dashed lines and the surrounding GFP-positive cells were used as controls. Nuclei were stained with DAPI (blue). N410. Bar, 20 μm. (e’) Line scan across the dPtpmeg mutant clone to show the relative fluorescent intensities of phosphotyrosine (pTyr) in control (GFP-positive) and homozygous dPtpmeg mutant (GFP-negative) cells. (f–h) Compared with wild-type wing (f), ectopic expression of wild-type dPtpmeg (g), but not the phosphatase-deficient mutant dPtpmeg-CS (h), in the posterior compartment of the wing using engrailed-Gal4 (en-Gal4) caused a wing vein missing phenotype. The dashed line indicates the boundary between anterior (a) and posterior (p) compartments. (i) Coexpression of dPtpmeg-RNAi rescued dPtpmeg-induced wing vein defects. (j) Ectopic expression of PTP61F did not affect wing vein pattern. (k–o) The dPtpmeg-induced wing vein defects could be suppressed by EgfrElp (k) and gap1 (l) mutants, or by the coexpression of RasV12 (m), but not by coexpressing the constitutively active phosphoinositide 3-kinase (Dp110-CAAX) (n) or active Akt (myr-Akt) (o). Genetic analyses were performed for three times with 100% penetrance of the phenotype (N4100 for each genotype). (p) Clonal expression of dPtpmeg (GFPpositive cells) in the larval wing imaginal discs resulted in a marked decrease in phospho-MAPK staining (blue) in L3 and L4 proveins. The dPtpmeg-overexpressing clones were marked with white dashed lines and the surrounding GFP-negative cells were used as controls. Actin was stained with tetramethylrhodamine isothiocyanate (TRITC)-labeled phalloidin (red). N410. Bar, 50 μm. (p’) High-magnification view of the area indicated by a white square in (p). Oncogene (2014), 1 – 13

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shown to induce Eps15 tyrosine phosphorylation.29 To test whether dPtpmeg would recognize Eps15 as a substrate in vivo, the full-length WT or DA mutant form of dPtpmeg was

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3 coexpressed with Eps15 in S2 cells for substrate trapping. As shown in Figure 2b, we found a significant amount of Eps15 to associate with the substrate-trapping DA mutant and substantially

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4 less with the WT form of dPtpmeg. Moreover, the tyrosine phosphorylation level of Eps15 was markedly increased in S2 cells treated with double-stranded RNA knocking down dPtpmeg (Figure 2c). These results suggest that dPtpmeg plays a critical role in regulating the function of Eps15 through direct tyrosine dephosphorylation. Eps15 is a substrate of PTPN3 in mammalian cells dPtpmeg is the Drosophila homolog of two closely related mammalian non-transmembrane protein tyrosine phosphatases: PTPN3 and PTPN4. It has been shown that PTPN4 is highly expressed in brain and plays an important role in motor learning and cerebellar plasticity.30,31 On the other hand, PTPN3 has been implicated in regulating cell growth and human tumorigenesis.17,32 Here we investigated whether Eps15 is a substrate of PTPN3 in mammalian cells. HEK293T cells were transiently transfected with hemagglutinin (HA)-tagged wild-type PTPN3 (PTPN3-WT) or substrate-trapping mutant PTPN3 (PTPN3-DA) or a PTP domain deleted PTPN3 (PTPN3-ΔPTP) with the Flag-tagged Eps15. Immunoblotting of the anti-HA immunoprecipitates from cell lysates revealed that Eps15 co-immunoprecipitated with the WT and DA mutant forms of PTPN3, but not with PTPN3-ΔPTP (Figure 2d). Eps15 has been shown to be phosphorylated at tyrosine residue 850 following EGFR activation.33 To determine whether Y850 is the recognition site of PTPN3, we checked whether PTPN3 can interact with the Eps15 tyrosine phosphorylation-defective mutant (Eps15-Y850F). Interestingly, neither PTPN3-WT nor PTPN3-DA could co-immunoprecipitate with Eps15-Y850F, suggesting that PTPN3 interacts with Eps15 in a phosphorylation-dependent mechanism. We next examined the effect of WT and various mutant forms of PTPN3 on the tyrosine phosphorylation levels of Eps15 with a pEps15 (Y850)-specific antibody. As shown in Figure 2e, EGF-induced tyrosine phosphorylation of Eps15 was markedly diminished when it was coexpressed with WT but not the catalytically inactive PTPN3 (PTPN3-CS) or PTPN3-ΔPTP. We also found that, unlike PTPN3, expression of WT or the CS mutant form of PTPN23 and Shp2 did not affect EGF-induced Eps15 tyrosine phosphorylation (Supplementary Figures S1B and C). To determine whether PTPN3 can directly interact with Eps15, in vitro pull-down assay was performed. As bacterial expression of the full-length Eps15 formed insoluble aggregates, we performed glutathione S-transferase (GST) pull-down assays with bacterially expressed Flag-Eps15ΔN (amino acids (aa) 590–897) and GST-PTPN3ΔN (aa 507–909, containing the PDZ and PTP domain) (Supplementary

Figure S1D). As shown in Figures 2f and g, bacterially expressed Flag-Eps15ΔN is tyrosine phosphorylated and interacts with both WT and DA forms of GST-PTPN3ΔN. We further confirmed the interaction using in vitro translated and phosphorylated FlagEps15 and bacterially expressed GST-PTPN3ΔN (Supplementary Figures S1E and F). Moreover, the role of PTPN3 on dephosphorylation of Eps15 was examined by in vitro dephosphorylation assay with bacterially expressed Flag-Eps15ΔN (aa 590–897) and GST-PTPN3ΔN. We found that WT, but not the substrate-trapping mutant (DA), form of GST-PTPN3ΔN promoted the dephosphorylation of pEps15 (Tyr 850) in a dosage-dependent manner (Figure 2h). These results together indicate that Eps15 is a substrate of mammalian PTPN3. PTPN3-mediated Eps15 dephosphorylation promotes EGFR for lysosomal degradation Endocytosis plays an important role in the regulation of EGFR activation and cell signaling.34 Upon EGF stimulation, activated EGFR can be internalized to various subcellular compartments, recycling or to lysosome for degradation. Tyrosine phosphorylation of Eps15 is known to play an important role in regulating EGFR endocytosis.33 To determine the role of PTPN3 in EGFR trafficking, we first checked endogenous PTPN3 localization by immunofluorescence under basal and EGF-stimulated conditions. PTPN3 displayed a diffuse cytosolic distribution with scattered small puncta at the periphery of H1975 and A549 lung cancer cells not treated with EGF (Figure 3a and Supplementary Figure S2A). When cells were stimulated with EGF for 5 min, PTPN3 became distributed in a distinct punctate pattern that colocalized with EGFR. Next, we examined whether PTPN3 could interact and dephosphorylate EGFR upon EGF stimulation. As shown in Supplementary Figure S1G, EGFR did not co-immunoprecipitate with either WT or DA mutant form of PTPN3. Moreover, expression of PTPN3-WT or PTPN3-CS did not cause any significant change of EGFR tyrosine phosphorylation at Y845, Y1045, Y1068, Y1148 and Y1173 tyrosine sites (Supplementary Figure S1H). Therefore, PTPN3 is more than likely involved in EGFR signaling specific through Eps15 in endosomal trafficking. To investigate whether PTPN3-mediated Eps15 dephosphorylation would affect EGFR internalization in response to EGF stimulation, lung cancer cells (H1975) stably expressing PTPN3 or Eps15 tyrosine phosphorylation-deficient mutant (Y850F) were treated with EGF-Alexa 488 to stimulate EGFR endocytosis. Quantitative internalization assay by flow cytometry showed that the rate of EGF-Alexa 488 internalization in PTPN3- and Eps15-Y850F-

Figure 2. Identification of Eps15 as a substrate for Drosophila Ptpmeg and human PTPN3. (a) Pervanadate-treated S2 cell lysates were incubated with either the WT or the DA trapping mutant form of HA-tagged dPtpmeg-PTP (catalytic domain of dPtpmeg). After immunoprecipitation using anti-HA antibody, dPtpmeg-PTP and its associated proteins were resolved by SDS–PAGE and stained with Sypro Ruby. The proteins associated with the DA mutant, but not the WT, form of dPtpmeg-PTP were excised for mass spectrometry analysis. The proteins identified in the dPtpmeg-trapping complexes are listed with protein score and numbers of peptides. (b) V5-tagged Eps15 was ectopically coexpressed with the HA-tagged WT form or the DA mutant form of dPtpmeg in S2 cells. After immunoprecipitation using anti-V5 antibody, the immunoprecipitates and total cell lysates (TCLs) were analyzed by immunoblotting with antibodies as indicated. (c) S2 cells treated with GFP double-stranded RNA (dsRNA; control) or dPtpmeg dsRNA were transfected with V5-tagged Eps15. Immunoprecipitated Eps15 was analyzed by immunoblotting with the indicated antibodies. The efficiency of dPtpmeg knockdown was verified by western blotting with dPtpmeg antibody (lower panel). The levels of phosphotyrosine (pTyr) were quantified with ImageJ (NIH, Bethesda, MD, USA) and normalized to the immunoprecipitated Eps15. Data are represented as mean ± s.d. from triplicate experiments. *P = 0.02. (d) HEK293T cells transfected with FLAG-tagged full-length Eps15 or Eps15-Y850F, together with HA-tagged PTPN3 WT, D811A (DA) or ΔPTP (PTP domain deletion mutant), were subjected to immunoprecipitations with anti-HA antibody. The immunoprecipitates and total cell lysates were analyzed by immunoblotting with antibodies as indicated. (e) Lysates of HEK293T cells transfected with FLAG-tagged full-length Eps15 WT or Y850F (YF), together with HA-tagged PTPN3 WT, C842S (CS) or ΔPTP, were immunoprecipitated with anti-FLAG antibody. The immunoprecipitates and cell lysates were analyzed by immunoblotting with antibodies as indicated. (f) Bacterially expressed Flag-Eps15ΔN is tyrosine phosphorylated at Tyr850. Equal amounts of bacterially expressed and purified Flag-Eps15ΔN WT and YF were analyzed by immunoblotting with antibodies as indicated. (g) Bacterially expressed Flag-Eps15ΔN was mixed with equal amounts of GST or GST-PTPN3ΔN WT or DA fusion proteins for in vitro pull-down assays. (h) PTPN3 dephosphorylates Eps15 in vitro. Purified GST (10 μg), GST-PTPN3ΔN DA (10 μg) or different amounts of GST-PTPN3ΔN (2, 5 and 10 μg) were incubated with FLAG-Eps15ΔN WT or YF in a PTPase buffer at 37 °C for 30 min. Numbers below lanes indicate the relative intensities of the pEps15 bands. Oncogene (2014), 1 – 13

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5 expressing cells was comparable to that of control cells (Figure 3b and Supplementary Table S1). We further performed immunofluorescence assays to determine the effect of PTPN3 and Eps15-

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Y850F on the intracellular localization of EGF-Alexa 488 and endosomal markers (EEA1 and Rab11) and the lysosomal marker (LAMP-2). In control cells, we found that 5 min after internalization,

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Figure 3. PTPN3-mediated Eps15 dephosphorylation accelerates sorting of EGFR for lysosomal degradation. (a) H1975 cells were treated without or with EGF (100 ng/ml) for 5 min and immunostained with anti-PTPN3 (red) and anti-EGFR (green) antibodies. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) in blue. The insets show a higher magnification of the area enclosed within the white box. Bar, 10 μm. (b) H1975 cells stably expressing HA-tagged PTPN3, FLAG-tagged Eps15-Y850F or an empty vector control were treated with EGF-Alexa 488 (100 ng/ml) for 1 h at 4 °C. Cells were then incubated at 37 °C for 5, 10 and 15 min for the internalization of EGF-Alexa 488. The rate of EGF internalization was determined by flow cytometry as described in the Materials and methods. Data represent the mean ± s.d. of three independent experiments. (c) H1975 cells stably expressing HA-tagged PTPN3, FLAG-tagged Eps15-Y850F or an empty vector control were treated with EGF-Alexa 488 (100 ng/ml) for 5 min. Ectopic expression of PTPN3 or Eps15-Y850F caused the colocalization of EGF-Alexa 488 and LAMP-2, compared with controls. Nuclei were stained with DAPI in blue. The insets show a higher magnification of the area enclosed within the white box. Bar, 10 μm. (d) Percentage colocalization of EGF-Alexa 488 with LAMP-2 in (c). Data are represented as mean ± s.d. of triplicates, with an average of 10 cells scored per experiment. ***Po 0.001. (e, f) H1975 cells stably expressing HA-tagged PTPN3, FLAG-tagged Eps15Y850F or an empty vector control were treated with 100 μM Bafilomycin A1 (Baf-A1) for 60 min, followed by incubation with 100 ng/ml EGF for the indicated times. Cell lysates were analyzed by immunoblotting with antibodies as indicated.

EGF is dispersed in the cytosol and colocalized with the early endosomal marker EEA1 (Supplementary Figures S3A and B). However, EGF largely colocalized with LAMP-2 in PTPN3- or Eps15Y850F-expressing cells as compared with controls (Figures 3c and d). Moreover, ectopic expression of PTPN3 in H1975 and A549 cells showed that WT PTPN3, but not the catalytically inactive mutant PTPN3-CS, resulted in a marked decrease in EGF-induced phosphorylation and activation of MAPK (Figure 3e and Supplementary Figures S2B and S3C), suggesting that, like Drosophila Ptpmeg, PTPN3 negatively regulates the EGFR/MAPK signaling pathway in lung cancer cells. Notably, PTPN3 overexpression also caused a drastic reduction in the EGFR protein levels upon EGF stimulation (Figure 3e). In addition, like PTPN3, expression of Y850F mutation but not wild-type Eps15 caused a similar reduction in the EGFR protein levels as well as EGF-induced phosphorylation of MAPK (Figure 3f). Eps15 has also been shown to regulate the endocytic trafficking of the hepatocyte growth factor receptor Met.35 The hepatocyte growth factor–Met signaling plays a critical role in the growth, invasion and metastasis of various human cancers.36,37 However, we noticed that PTPN3 and Eps15-Y850F mutant did not affect Met receptor levels after Oncogene (2014), 1 – 13

hepatocyte growth factor stimulation (Supplementary Figure S4), suggesting that PTPN3-Eps15 plays a specific role in regulating EGFR signaling. We next investigated whether the PTPN3 and Eps15-Y850F-induced EGFR degradation could be blocked by treating cells with a proteasomal inhibitor (MG132) or a lysosomal inhibitor (bafilomycin A1). Treatment of cells with bafilomycin A1 but not MG132 suppressed PTPN3- and Eps15-Y850F-induced degradation of EGFR (Figures 3e and f and Supplementary Figures S5A and B). Together, these results indicate that PTPN3-mediated Eps15 dephosphorylation promotes the ligand-bound EGFR for lysosomal degradation and downregulates EGFR signaling. PTPN3 promotes EGFR degradation via non-clathrin-mediated endocytosis In addition to the clathrin-mediated endocytosis, several studies have reported that EGFR can also be internalized via caveolae/ lipid raft-mediated endocytosis, a clathrin-independent pathway.38 Clathrin-mediated endocytosis has been shown to play a major role for sustained EGFR signaling, whereas non-clathrinmediated endocytosis was shown to be preferentially for EGFR © 2014 Macmillan Publishers Limited

PTPN3 negatively regulates EGFR signaling M-Y Li et al

degradation,39 although the molecular mechanisms in regulating these two internalization processes remained unclear. Interestingly, Eps15 is reported to be involved in both clathrin-dependent and -independent endocytosis of the EGFR.38,40 To investigate whether PTPN3-mediated Eps15 dephosphorylation plays a role in partitioning EGFR to non-clathrin-mediated endocytosis for degradation, we used confocal microscopy to examine the localization of EGFR, clathrin and caveolin-1 in control, PTPN3or Eps15-Y850F-expressing H1975 cells. As shown in Supplementary Figure S6, internalized EGF colocalized with clathrin in control but not in PTPN3- or Eps15-Y850Foverexpressing cells (Supplementary Figures S6A and C). Notably, internalized EGF colocalized with caveolin-1 in PTPN3- or Eps15Y850F-overexpressing cells, but the controls did not (Supplementary Figures S6B and D). To further confirm our observation, we used the sucrose density gradient fractionation approach to quantify the distribution of EGFR between clathrinenriched compartments and caveolin-1 lipid rafts in control, PTPN3- or Eps15-Y850F-expressing cells. The caveolin-enriched membranes (lipid raft) contained high levels of cholesterol and sphingolipids and migrated in the low-density region (fractions 3–5) of the sucrose gradient, whereas clathrin heavy chain was localized in the high-density non-lipid raft fractions (fractions 8–10) (Figure 4). In control H1975 cells, EGFR was enriched in the non-lipid raft fractions (Figure 4a). However, a substantial fraction of EGFR was found in the caveolin-enriched fractions in PTPN3and Eps15-Y850F-overexpressing cells (Figures 4b–d). Finally, we examined whether PTPN3- or Eps15-Y850F-induced EGFR degradation could be affected by treating cells with methyl-βcyclodextrin (MβCD) and filipin that are known to disrupt the integrity of membrane lipid rafts.41,42 As shown in Figures 4e and f and Supplementary Figures S7A and B, the degradation of EGFR was dramatically reduced in the presence of MβCD and filipin.

7 Together, these results suggest that PTPN3-mediated Eps15 dephosphorylation directs EGFR to lipid raft-mediated endocytosis for degradation. Ectopic expression of PTPN3 and Eps15-Y850F inhibits lung cancer growth Lung cancer is the leading cause of cancer-related deaths worldwide. Accumulating evidence has shown that EGFR gene is often amplified or mutated within the tyrosine kinase domain in NSCLC.43,44 Because recent studies indicated an involvement of PTPN3 in lung tumorigenesis,45 we examined the role of PTPN3Eps15 in EGFR-dependent lung cancer growth. As shown in Figures 5a and b, ectopic expression of PTPN3 or Eps15-Y850F markedly inhibited cell proliferation in WST-1 conversion assay and colony formation assay compared with controls. We next tested whether PTPN3 and Eps15-Y850F may have an effect on lung cancer cell migration using the in vitro wound-healing and transwell migration assays. H1975 cells expressing PTPN3 or Eps15-Y850F migrated across the wound much slower than that of control cells over 24 h (Figure 5c). In the transwell assay, compared with control cells, there was a significant decrease in the number of PTPN3- or Eps15-Y850F-expressing cells that migrated across the transwell membrane to the underside of the inserts (Figure 5d). These results indicate that PTPN3 and Eps15-Y850F possess the potential for tumor suppression. To investigate whether PTPN3 and Eps15-Y850F have a growth inhibitory effect on tumor formation and development in vivo, we subcutaneously injected H1975 cells stably expressing PTPN3, Eps15-Y850F or vector only controls into athymic nude mice and measured tumor volume over time. As shown in Figure 5e, overexpression of PTPN3 or Eps15-Y850F caused a marked decrease in the tumor growth rate (Figure 5e). We further examined the expression level of EGFR

Figure 4. PTPN3-mediated Eps15 dephosphorylation promotes EGFR endocytotic trafficking through a non-clathrin pathway. (a–c) Lysates of H1975 cells expressing an empty vector control, PTPN3, or Eps15-Y850F were fractionated by sucrose density gradient centrifugation, and aliquots were immunoblotted with anti-EGFR, anti-clathrin and anti-caveolin-1 antibodies. Fractions 3–5 were enriched with caveolin-1 and denoted as lipid raft fractions, whereas fractions 8–10 were enriched with clathrin and denoted as non-lipid raft fractions. (d) Quantitative analysis for the relative distribution of EGFR in lipid raft fractions and non-lipid raft fractions. Data are represented as mean ± s.d. of triplicates. *P o 0.05. (e, f) H1975 cells stably expressing HA-tagged PTPN3, Eps15-Y850F or an empty vector control were treated with 100 μM MβCD for 60 min, followed by incubation with 100 ng/ml EGF for the indicated times. Cell lysates were analyzed by immunoblotting with antibodies as indicated. © 2014 Macmillan Publishers Limited

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Figure 5. PTPN3 and Eps15-Y850F negatively regulate cell proliferation of NSCLC in vitro and tumor growth in vivo. (a) A time course of cell proliferation by WST-1 assay of H1975 cells expressing PTPN3 (red line), Eps15-Y850F (green line) or vector only control (blue line). Data are represented as mean ± s.d. of triplicates. (b) Colony formation assay of H1975 cells stably expressing PTPN3, Eps15-Y850F or an empty vector control. The colonies were stained with crystal violet and counted. Data are represented as mean ± s.d. of triplicates. (c) H1975 cells stably expressing PTPN3, Eps15-Y850F or an empty vector control were cultured to confluent monolayer, wounded and incubated in growth medium containing 10 μg/ml mitomycin. Cell migration was observed with light microscope at indicated time points. (d) Cell migration was also assessed using in vitro transwell migration assay. Cells in (c) were plated onto the upper well of Transwell Boyden chamber and allowed to migrate for 24 h. Cells that migrated through the filter were stained with crystal violet and quantified in a microplate reader. Data are represented as mean ( ± s.d.) value of migrated cells (n = 3). (e) H1975 cells infected with PTPN3, Eps15-Y850F or vector only control were injected subcutaneously into the right and left sides, respectively, of the flank region of nude mice. Tumor volume was monitored for the indicated times (0, 7, 14, 21 and 28 days). Data are represented as mean ± s.d. of triplicates. (f) Immunohistochemical analysis of EGFR and pEps15 (Y850) in tumor xenografts from (e). Bar, 200 μm. *P o0.05; ***P o0.001.

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9 and phospho-Eps15 in tumors harvested 4 weeks after injection. Immunohistochemical staining showed that PTPN3 and Eps15Y850F drastically reduced the amount of EGFR and phosphoEps15 in excised tumors (Figure 5f). Together, these results suggest that PTPN3-mediated Eps15 dephosphorylation inhibits lung cancer formation and progression in vivo. Depletion of PTPN3 impairs EGFR degradation and enhances the tumorigenic potential of lung cancer cells We next examined whether ablation of PTPN3 would enhance the oncogenic properties of lung cancer cells. Quantitative realtime–PCR analyses revealed higher expression of PTPN3 in CL1-5 cells compared with other NSCLC cell lines (H520, H1975, A549, H1299 and H928) (Supplementary Figure S8A). Two different sets of small hairpin RNAs (shRNAs) that can effectively downregulate the expression of PTPN3 were stably expressed in CL1-5 human lung cancer cells (Supplementary Figure S8B). Intriguingly, immunoblot analysis showed that depletion of PTPN3 caused an increase in the level of EGFR and EGFR-mediated MAPK phosphorylation (Figure 6a). After 24 h of EGF stimulation, the level of EGFR expression decreased in scrambled shRNA knockdown control cells (Figure 6b), but not in PTPN3 knockdown cells. Consistent with our earlier observations, shRNA-mediated knockdown of PTPN3 caused a marked increase of Eps15 p-Tyr 850 levels but there was no significant change of EGFR tyrosine phosphorylation at Y845, Y1045, Y1068, Y1148 and Y1173 sites (Figures 6c and d). We further checked whether PTPN3 depletion might affect EGFR endocytic trafficking by immunofluorescence assay. At 5 min after stimulation, depletion of PTPN3 did not affect EGFR distribution or the endocytic pathway, compared with controls (Figure 6e and Supplementary Figures S8C and D). After 60 min of stimulation, EGF-Alexa 488 largely colocalized with LAMP-2 in control cells (Figure 6f). However, EGF was dispersed in the cytoplasm and did not significantly colocalize with LAMP-2 in PTPN3 knockdown cells. Moreover, we found that PTPN3 knockdown resulted in a substantial increase in CL1-5 lung cancer cell proliferation and cell growth, as observed by WST-1 and colony formation assay, respectively (Figures 7a and b). The in vivo effect of PTPN3 on tumor growth was examined in a xenograft model in nude mice. CL1-5 cells expressing shPTPN3 or a scrambled shRNA control were subcutaneously injected into athymic nude mice and tumor volume was measured over time. Compared with controls, depletion of PTPN3 significantly increased the tumor growth rate (Figure 7c). Immunohistochemical staining showed that depletion of PTPN3 led to an increase in EGFR and phospho-Eps15 levels in excised tumors (Figure 7d). Considered together, these results demonstrate that PTPN3 indeed acts as a negative regulator of EGFR signaling and EGFR-mediated lung cancer growth. DISCUSSION The EGFR belongs to the ErbB family of protein tyrosine kinases and is a major regulator for both normal development and cancer progression.46 PTPs, which include receptor-like PTPs and nonreceptor PTPs, are a group of tightly regulated enzymes thought to regulate tyrosine phosphorylation by antagonizing the action protein tyrosine kinases.1,10 In this study, we provide the first evidence that PTPN3 inhibits the EGFR signaling by targeting the receptor for lysosomal degradation. In Drosophila, dPtpmeg antagonizes receptor tyrosine kinase activity and plays a role in controlling border cell migration during oogenesis. Moreover, dPtpmeg negatively regulates the EGFR/Ras/MAPK pathway during wing morphogenesis. Substrate-trapping and biochemical analysis further identified Eps15 as a substrate for dPTPmeg and PTPN3. Our results demonstrate that PTPN3 dephosphorylates Eps15 and promotes EGFR for degradation in lung cancer cells. © 2014 Macmillan Publishers Limited

Eps15 is a multidomain adaptor protein that plays an important role in regulating endocytic trafficking. In mammalian cells, Eps15 can be phosphorylated by EGFR at tyrosine residue 850 upon EGF stimulation.29,33 Confalonieri et al.33 showed that ectopic expression of Eps15-Y850F mutant impairs the internalization of EGFR. On the contrary, using immunofluorescence and flow cytometry, we showed that ectopic expression of PTPN3 and Eps15-Y850F did not affect the internalization of EGFR. As we found a dramatic reduction of EGFR levels in cells expressing PTPN3 and Eps15-Y850F, one explanation for our results being contradictory to previous findings33 might be because of the difference in detection sensitivity. In addition to its role in endocytic trafficking, Eps15 was reported to localize at the trans-Golgi network and regulate vesicle trafficking during the secretory process.47 However, immunofluorescence analysis of the intracellular distribution of endogenous PTPN3 or exogenously expressed HA-tagged PTPN3 indicated no significant localization at the trans-Golgi network (data not shown), and this might suggest that PTPN3 is not involved in Eps15-mediated protein sorting and vesicle trafficking at the trans-Golgi network. The ligand-activated EGFR and the transforming growth factorβ receptor have been reported to be endocytosed through a clathrin-dependent as well as a clathrin-independent pathway.39,48 Segregation of these cell surface receptors through distinct endocytic pathways is known to regulate downstream signal duration and receptor trafficking, although it is unclear how it does this. Several lines of evidence indicate that PTPN3 and Eps15-Y850F accelerate downregulation of EGFR via a clathrinindependent but lipid raft-dependent pathway. First, EGF-488 trafficking assay revealed that EGF-488 was largely colocalized with lipid raft-associated protein caveolin-1 but not with clathrin in cells expressing PTPN3 or Eps15-Y850F (Supplementary Figure S6). Second, analysis of EGFR profile by sucrose gradient fractionation showed that EGFR was concentrated in clathrin-enriched non-lipid fractions in control cells (Figure 4a). However, overexpression of PTPN3 and Eps15-Y850F led to a redistribution of EGFR to caveolin-1-enriched lipid raft fractions (Figures 4b–d). Third, disruption of lipid rafts with MβCD and filipin suppressed PTPN3- or Eps15-Y850F-induced EGFR degradation (Figures 4e and f). How does PTPN3-mediated tyrosine dephosphorylation of Eps15 regulate EGFR for lipid raft-dependent endocytosis and lysosomal degradation? Accumulating evidence has shown that EGFR ubiquitination is not essential for its internalization, but appears to play an important role in endosomal sorting and lysosomal targeting of the receptor.49,50 One possibility is that tyrosine dephosphorylation of Eps15 by PTPN3 may affect the ubiquitination status of EGFR, accelerating EGFR for lysosomal degradation. Recently, Roxrud et al.51 have identified an endosomally localized Eps15 isoform (Eps15b) that interacts with the Hrs (hepatocyte growth factor–regulated tyrosine kinase substrate) complex to mediate EGFR degradation. We found that like Eps15, Eps15b is also a substrate of PTPN3 (M-Y Li and G-C Chen, unpublished data), indicating a dual role for PTPN3 in the regulation of endocytic trafficking and endosomal sorting of EGFR. Accumulating evidence has indicated that PTPs can function as tumor suppressors or oncogenes depending on the substrate involved and the cellular context.32,52 It has been reported that PTPN3 expresses in gastric cancer cells and may play a role in gastric cancer progression and differentiation53 PTPN3 has been found to coordinate with p38γ MAPK to promote Ras oncogenesis in colon cancer,54 and it has been found to stimulate breast cancer growth by inducing and stabilizing the protein expression of vitamin D receptor.55 Interestingly, recent studies have also indicated that PTPN3 plays a role in tumor suppression. Mutational analysis of the tyrosine phosphatome found PTPN3 along with five other PTPs (PTPRF, PTPRG, PTPRT, PTPN13 and PTPN14) to be Oncogene (2014), 1 – 13

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mutated in colorectal cancer.56 Moreover, Jung et al.45 analyzed the transcriptome of two NSCLC cell lines and found that one allele of PTPN3 is mutated in the NSCLC cell line H2228. They further showed that ectopic expression of PTPN3 inhibits the growth of NSCLC cells, although the molecular mechanisms underlying the growth inhibition remain unknown. Our

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data support a model in which PTPN3-mediated tyrosine dephosphorylation of Eps15 leads to EGFR degradation and tumor suppression in NSCLC cells. This study demonstrated that PTPN3 and Eps15-Y850F overexpression reduced EGFR protein levels and impeded the proliferation and migration of NSCLC cells. Moreover, PTPN3 and Eps15-Y850F significantly suppressed NSCLC tumor

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11 growth in a subcutaneous xenograft model. Conversely, depletion of PTPN3 enhanced EGFR stabilization and promoted NSCLC tumorigenicity both in vitro and in vivo. Our findings are consistent with the idea that PTPN3 acts as a tumor suppressor in NSCLC. In conclusion, we have identified Eps15 as an evolutionarily conserved dPtpmeg/PTPN3 substrate that regulates EGFR signaling. The finding that PTPN3-mediated tyrosine dephosphorylation of Eps15 modulates EGFR-dependent cancer progression may

help contribute to the development of a targeting intervention in NSCLC. MATERIALS AND METHODS For additional information concerning Drosophila genetics, immunofluorescence staining and cell migration assays, please refer to Supplementary Information.

Figure 7. Depletion of PTPN3 promotes cell proliferation of NSCLC in vitro and tumor growth in vivo. (a) A time course of cell proliferation by WST-1 assay of CL1-5 cells infected with scramble (blue line), shPTPN3-A1 (green line) or shPTPN3-D1 (red line). (b) Colony formation assay of CL1-5 cells infected with scramble control or shPTPN3 (A1 and D1). The colonies were stained with crystal violet and counted. Data are represented as mean ± s.d. of triplicates. (c) CL1-5 cells infected with scramble control or shPTPN3-A1 were subcutaneously injected into the right and left flank region of athymic nude mice. Tumor volume was monitored for the indicated times (0, 7, 14 and 21 days). Data are represented as mean ± s.d. of triplicates. (d) Immunohistochemical analysis of EGFR and phospho-Eps15 (Y850) in tumor xenografts from (c). Bar, 200 μm. *Po 0.05; ***Po 0.001.

Figure 6. Depletion of PTPN3 leads to an impairment of EGFR degradation. (a) CL1-5 cells infected with scramble control, shPTPN3-A1 or shPTPN3-D1 were treated with EGF (100 ng/ml) for the indicated times (0, 5, 15 and 30 min). Cell lysates were analyzed by immunoblotting with antibodies as indicated. (b) CL1-5 cells infected with scramble control, shPTPN3-A1 or shPTPN3-D1 were treated with cycloheximide and EGF (100 ng/ml) for the indicated times (0, 6, 12 and 24 h). Cell lysates were analyzed by immunoblotting with antibodies as indicated. (c) CL15 cells infected with scramble control, shPTPN3-A1 or shPTPN3-D1 were stimulated with EGF (100 ng/ml) for 5 min and cell lysates were analyzed by immunoblotting with antibodies as indicated. (d) CL1-5 cells infected with scramble control, shPTPN3-A1 or shPTPN3-D1 were stimulated with EGF (100 ng/ml) for 5 min and cell lysates were immunoprecipitated with anti-EGFR antibody. The immunoprecipitates were analyzed by immunoblotting with pEGFR antibodies. (e, f) CL1-5 cells infected with scramble control, shPTPN3-A1 or shPTPN3-D1 were treated with EGF-Alexa 488 (100 ng/ml) for 5 min (e) or 60 min (f) and immunostained with anti-LAMP-2 antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) in blue. The insets show a higher magnification of the area enclosed within the white box. Bar, 10 μm. Percentage colocalization of EGF-Alexa 488 with LAMP-2 is shown in the bottom panels. Data are represented as mean ± s.d. of triplicates, with an average of 10 cells scored per experiment. ***Po 0.001. © 2014 Macmillan Publishers Limited

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12 Plasmids and antibodies Human PTPN3 complementary DNA was obtained from Open Biosystems (Thermo, Waltham, MA, USA). HA-tagged PTPN3 WT, DA (D811A), CS (C842S) and ΔPTP (PTP domain deletion mutant, lacking residues 611 to 913) were generated by PCR and subcloned into pcDNA and pSin-EF2-Pur lentiviral vector. Flag-Eps15-Wt and Flag-Eps15-Y850F were kind gifts from Pier Paolo Di Fiore (IEO- European Institute of Oncology, Milan, Italy). The lentiviral shRNA clones used to knock down human PTPN3 were obtained from the National RNAi Core Facility of Academia Sinica (Taipei, Taiwan). The targeted sequences for these clones are PTPN3 shRNA #A: (clone ID: TRCN0000002788) 5′-CGTGTGTATGAAGAAGGTTTA-3′ and PTPN3 shRNA #D: (clone ID: TRCN0000320903) 5′-CCAAGAGAGTTTATCCGAGAA-3′. Scramble shRNA was used as a control (Addgene, Cambridge, MA, USA). Lentiviral production and infection were performed as previously described.57 Antibodies used for the study were: anti-pMAPK (Sigma, St Louis, MO, USA), anti-MAPK (Sigma), anti-LAMP-2 (Abcam, Cambridge, UK), anti-EEA1 (Cell Signaling, Danvers, MA, USA), anti-LAMP1 (Abcam), anti-Met (Abcam), anti-EGFR and anti-phospho EGFR antibodies (Cell Signaling), anti-fascin (Developmental Studies Hybridoma Bank, Iowa City, IA, USA). Anti-PTPN3 antibody was a generous gift from Nicholas Tonks (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA). Anti-phospho Eps15 (Y850) antibody was generated by immunizing rabbits with a synthetic phosphopeptide (FANFSApYPSEEDMIC, bovine serum albumin coupled) corresponding to residues surrounding Y850 of mouse Eps15. AntidPtpmeg antibody was generated by immunizing rabbits with a synthetic peptide (RKPANAPKNRYRDISPYDC, ovalbumin coupled) corresponding to residues of Drosophila dPtpmeg.

Cell culture, immunoprecipitation and immunoblotting Drosophila S2 cells were cultured at 25 °C in Schneider’s Drosophila medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum and 1 × penicillin/streptomycin antibiotics (Invitrogen). HEK293T cells were cultured at 37 °C in DMEM (Invitrogen) supplemented with 10% fetal bovine serum and antibiotics. H1975 and CL1-5 lung cancer cells were grown in RPMI-1640 (Invitrogen) with 10% fetal bovine serum. For immunoprecipitations, cells transiently transfected with the indicated plasmids were scraped from dishes in lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1 mM EDTA, 10 mM NaF, 2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (Roche, Indianapolis, IN, USA)). Cell lysates were immunoprecipitated with anti-FLAG or anti-V5 antibody and protein G-Sepharose beads (GE Healthcare, Piscataway, NJ, USA) at 4 °C for 3 h. These beads were washed three times with the lysis buffer. After resolution by SDS–PAGE, the immunoprecipitates were subjected to western blot analysis.

In vitro pull-down assays GST-tagged WT or DA form of PTPN3ΔN (aa 507–909) (gift of Kai-En Chen, Academia Sinica, Taipei, Taiwan) and GST-tagged WT or YF form of FLAGEps15ΔN (aa 590–897) were purified from BL21 bacteria and eluted from Glutathione-Sepharose 4B beads (GE Healthcare) as suggested by the manufacturer. To remove the GST tag, GST-FLAG-Eps15ΔN was incubated with PreScission Protease (GE Healthcare) at 4 °C for 4 h. For binding assays, FLAG-Eps15ΔN was incubated with equivalent amounts of GST or GST-PTPN3ΔN fusion proteins and immobilized on glutathione beads. Following incubation, bead-bound proteins were separated with SDS– PAGE and analyzed by western blot analyses.

In vitro dephosphorylation assay Bacterially expressed FLAG-Eps15ΔN was incubated with GST and GSTPTPN3ΔN WT or GST-PTPN3ΔN DA proteins at 37 °C for 30 min with in vitro PTPase buffer (25 mM Tris-HCl, pH 7.5, 2.5 mM EDTA, 1 mM EGTA, 5 mM dithiothreitol, 1 mg/ml bovine serum albumin, 1 mM PMSF and protease inhibitor cocktail (Roche)). Reactions were stopped by adding sample buffer and boiling for 5 min. Samples were resolved by SDS–PAGE and subjected to western blot analysis.

Preparation of lipid raft membranes Lipid raft isolation was carried out by sucrose density gradient ultracentrifugation according to the method previously described.58 Briefly, harvested cells were resuspended in 1 ml of ice-cold 2-(N-morpholino) ethanesulfonic acid (MES) buffer saline (25 mM MES, pH 6.0, 150 mM NaCl, Oncogene (2014), 1 – 13

0.5% Triton X-100, 1 mM PMSF and protease inhibitor cocktail (Roche)) and homogenized by passing 10 times through a 27-gauge needle. The supernatant was mixed with 1 ml of 80% sucrose in MES, and overlaid with 5 ml of 35% sucrose in MES followed by 3 ml of 5% sucrose in MES. The samples were then centrifuged at 35 000 r.p.m. in a SW41Ti rotor (Beckman, Brea, CA, USA) for 20 h at 4 °C. The sucrose gradients were harvested in 1 ml fractions from the top of the gradient, and individual fractions were analyzed by western blotting.

Xenograft tumorigenicity assay Xenograft tumorigenicity assay was performed as previously described.59 Briefly, virus-infected H1975 and CL1-5 cells were harvested, washed with phosphate-buffered saline and resuspended in RPMI-1640 medium. Cells (1 × 106) were then injected subcutaneously into the right and left side, respectively, of the flank region of 8-week-old male BALB/c nude mice (Rodent Model Resource Center, Taipei, Taiwan). Tumors were measured with calipers every 7 days after injection. All mice were killed 28 days after injection, and tumors were surgically excised, weighed and photographed. Differences in tumor progression were statistically analyzed using Student’s t-test.

Statistical analysis All experiments have been repeated for at least three times. Statistical analysis was performed by Student’s t-test. Differences were considered significant if P-values were o 0.05 (*), 0.01 (**) and 0.001 (***).

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We thank Drs J Borst, PP Di Fiore, M McNiven, N Tonks, H Sun, KE Chen, the Bloomington Stock Center and Fly Core Taiwan for reagents. We are grateful to Dr J Settleman for helpful comments on the manuscript, Dr C-C Hung for confocal microscopy assistance and Y-L Huang for peptide synthesis. This work was supported by the National Science Council of Taiwan (NSC102-2311-B-001-027-MY3) and Academia Sinica.

AUTHOR CONTRIBUTIONS G-CC, M-YL and C-WW conceived and designed the experiments. M-YL, Y-TC, P-LL, A-PC, Y-ZM and G-DC conducted the experiments. G-CC, M-YL, K-HK and T-CM analyzed the data. G-CC and M-YL wrote the paper.

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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

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