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Protein Tyrosine Phosphatase PTPN3 Inhibits Lung Cancer Cell Proliferation and Migration by Promoting EGFR Endocytic Degradation

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Protein Tyrosine Phosphatase PTPN3 Inhibits Lung Cancer Cell Proliferation and Migration by Promoting EGFR Endocytic Degradation Oncogene (2015) 34, 3791–3803 © 2015 Macmillan Publishers Limited All rights reserved 0950-9232/15 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 (2015) 34, 3791–3803; doi:10.1038/onc.2014.312; published online 29 September 2014 INTRODUCTION sorting EGFR to multivesicular bodies.15 Recently, Ali et al.16 Reversible tyrosine protein phosphorylation by protein tyrosine showed that the ESCRT accessory protein HD-PTP/PTPN23 kinases and protein tyrosine phosphatases (PTPs) acts as a coordinates with the ubiquitin-specific peptidase UBPY to drive molecular switch that regulates a variety of biological pro- EGFR sorting to the multivesicular bodies. A better understanding cesses.1,2 The receptor tyrosine kinase epidermal growth factor of the role of PTPs in regulating EGFR signaling will help to receptor (EGFR), the best characterized member of the ErbB family provide insights into the molecular mechanisms behind EGFR- receptors, acts as a critical regulator of numerous cellular mediated tumorigenesis. processes, including growth, proliferation and differentiation. PTPN3 (PTPH1) and the closely-related PTPN4 (PTPMEG) are Upon activation by its growth factor ligands, EGFR undergoes non-transmembrane PTPs that contain an N-terminal FERM dimerization and activation, leading to tyrosine phosphorylation (Band 4.1, Ezrin, Radixin, Moesin homology) domain followed by of the intracellular region of the receptor as well as many a single PDZ (PSD95, Dlg, ZO1) domain and the C-terminal PTP 3,4 cytoplasmic substrates. The activated EGFR is then internalized domain.1 They have been implicated in the regulation of cell by clathrin-mediated endocytosis and sorted into the endosomal growth and proliferation.17,18 However, their role in receptor compartments, through which it is either recycled back to the protein tyrosine kinase signaling is not clear. The dPtpmeg is the plasma membrane or transported to the lysosome for degrada- 5 Drosophila homolog of mammalian PTPN3 and PTPN4. Phenotypic tion. Because overexpression or constitutive activation of EGFR analyses have revealed that dptpmeg mutants exhibit aberrant has been implicated in the pathogenesis and progression of a mushroom body axon projection patterns in the brain.19 Besides variety of human malignancies,6 it is therefore crucial to under- its role in regulating neuronal wiring, the molecular function of stand how EGFR signaling is regulated. dPtpmeg has remained largely unknown. In this study, we Several PTPs have been implicated in the regulation of EGFR fi signaling. Among them, PTPRK, DEP-1 (PTPRJ), PTP1B (PTPN1), identi ed EGFR pathway substrate 15 (Eps15) as a substrate of SHP-1 (PTPN6), TCPTP (PTPN2), PTPN9 and PTPN12 have been dPtpmeg and PTPN3. Eps15 is known to be an endocytic adaptor fi 20 shown to downregulate EGFR signaling by dephosphorylating involved in the regulation of EGFR traf cking. PTPN3 depho- EGFR.7–11 The receptor-type PTP DEP-1 dephosphorylates EGFR on sphorylated Eps15 and promoted EGFR for lipid raft-mediated the cell surface and inhibits its internalization.7 On the other hand, endocytosis and lysosomal degradation. The ectopic expression of the endoplasmic reticulum-localized PTP1B has been reported to PTPN3 or Eps15-Y850F mutant in the non-small-cell lung cancer regulate EGFR signaling from endosomes.12,13 PTP1B promotes (NSCLC) cells inhibited cell proliferation, migration and tumor the sequestration of EGFR onto internal vesicles of multivesicular growth. Our findings uncover a novel role for PTPN3 in the bodies.14 The ESCRT (endosomal sorting complex required for regulation of EGFR endocytic trafficking, degradation and transport) complexes are known to play an important role in signaling. 1Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; 2Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan and 3Institute 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; published online 29 September 2014 PTPN3 negatively regulates EGFR signaling M-Y Li et al 3792 RESULTS the activated form of Drosophila Ras (RasV12) (Figures 1k–m), but Drosophila Ptpmeg is involved in regulating the EGFR signaling not by coexpressing the constitutively active phosphoinositide 3- pathway kinase (Dp110-CAAX) or active Akt (myr-Akt) (Figures 1n–o). EGFR We previously performed genetic analyses to identify non- is known to induce extracellular signal-regulated kinase/MAPK transmembrane PTPs that could modulate border cell migration phosphorylation in future vein regions during wing develop- 21 ment.27 Strikingly, we found that clonal expression of dPtpmeg in during Drosophila oogenesis. Although RNA interference (RNAi)- 28 mediated downregulation of Drosophila non-transmembrane PTPs wing imaginal discs using the flipout/Gal4 system led to a did not have an obvious effect on border cell migration at stage marked reduction of MAPK phosphorylation in GFP-positive 10 egg chambers,21 we found that dptpmeg mutation caused dPtpmeg-expressing cells (Figures 1p, p’). Taken together, these accelerated migration of border cells and the clusters reached data indicate that dPtpmeg acts as a negative regulator of the oocyte prematurely at stage 9 (Figures 1a and b). The receptor EGFR/Ras/MAPK signal pathway. tyrosine kinases, platelet-derived growth factor (PDGF)- and vascular endothelial growth factor (VEGF)-related receptor (PVR) Identification of Eps15 as a substrate of dPtpmeg and EGFR, have been shown to play an important role in guiding 22,23 We have recently established a mass spectrometry (MS)-based migration of the border cells toward the oocyte. As shown in substrate trapping strategy to identify putative substrates of Figures 1c and d, ectopic expression of dPtpmeg with the border 26 fi PTP61F, the smallest non-transmembrane PTP in Drosophila. We cell-speci c Slbo-Gal4 driver impaired border cell motility. More- used the same approach to identify potential substrates of over, clonal analysis revealed elevated phosphotyrosine levels in dPtpmeg. The bacterially expressed wild-type (WT) form and the dptpmeg homozygous mutant border cell clones (marked by the substrate-trapping DA mutant form of dPtpmeg were incubated loss of green fluorescent protein (GFP); Figure 1e, e’), suggesting with Drosophila S2 cell lysates. After immunoprecipitation of that dPtpmeg may antagonize receptor tyrosine kinase-mediated dPtpmeg, the associated proteins were eluted, resolved by sodium tyrosine phosphorylation and border cell migration. dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) It has been reported that EGFR signaling regulates a variety of developmental processes in Drosophila, including wing vein and subjected to liquid chromatography tandem mass spectro- formation.24,25 To investigate the role of dPtpmeg in vein metry analysis. Several proteins, including Eps15, Pvr, Cortactin and Ter94 (mammalian VCP/p97), were identified to interact patterning, we used engrailed-Gal4 (en-Gal4) driver to restrict fi dPtpmeg expression in the posterior compartment of the wing; speci cally with the dPtpmeg-DA mutant (Figure 2a). Interestingly, fi therefore, the anterior part served as control. Interestingly, previous work has identi ed VCP/p97 as a substrate of PTPN3 in 17 fi expression of wild-type dPtpmeg, but not phosphatase-deficient mammalian cells. Moreover, the identi cation of Pvr as a fi mutant (dPtpmeg-CS), caused a wing vein missing phenotype putative substrate of dPtpmeg is consistent with the nding that (Figures 1f–h). The dPtpmeg-induced wing vein defects could be dPtpmeg plays a role in regulating border cell migration rescued by coexpressing dPtpmeg-RNAi (Figure 1i). Moreover, (Figures 1a–d). Here we have focused our study on Eps15. Eps15 ectopic expression of PTP61F,26 the Drosophila homolog of PTP1B, is a multidomain adaptor protein that contains EH domains at the in the developing
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