title: Identification of the amyloid β-protein precursor and cystatin C as novel epidermal growth factor receptor regulated secretory proteins in nasopharyngeal carcinoma by proteomics.
abstract: The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, while EGFR-modulated intracellular proteins have been extensively studied, little is known concerning their extracellular counterparts. To identify EGFR-regulated secreted proteins in NPC, we compared the secretome profiles of TGF-α-stimulated and unstimulated NPC cell line CNE-2. CNE-2 cells were cultured in the absence or presence of TGF-α for 24 h, and secreted proteins were obtained from conditioned serum-free media and enriched by ultrafiltration centrifugation. Using 2-DE and subsequent mass spectrometry, we identified 16 differential secreted proteins, among which the amyloid β-protein precursor (APP) was up-regulated and cystatin C was down-regulated after TGF-α stimulation. We further showed that the secretory changes of APP and cystatin C in CNE-2 after TGF-α stimulation could be abrogated by pretreatment of EGFR tyrosine kinase inhibitor PD153035 and PI3 kinase inhibitor Wortmannin, validating that APP and cystatin C are EGFR-regulated secreted proteins in NPC cells. Immunohistochemistry showed that the expression level of EGFR was positively correlated with the expression level of APP and negatively correlated with the expression level of cystatin C in NPC tissues, indicating that EGFR also regulates expression of APP and cystatin C in clinical NPC tissues. Furthermore, functional analysis showed that the growth and migration of CNE-2 cells decreased after neutralization of secretory APP in the medium using the anti-APP antibody. Our data provide substantial evidence that APP and cystatin C are target secreted proteins of EGFR in NPC, and upregulation of secretory APP by EGFR may be involved in the pathogenesis of NPC.
authors.affiliation: Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha 410008, China.
authors.forename: Can-E
authors.initials: CE
authors.lastname: Tang
authors.forename: Yong-Jun
authors.initials: YJ
authors.lastname: Guan
authors.forename: Bin
authors.initials: B
authors.lastname: Yi
authors.forename: Xin-Hui
authors.initials: XH
authors.lastname: Li
authors.forename: Ke
authors.initials: K
authors.lastname: Liang
authors.forename: Hai-Yan
authors.initials: HY
authors.lastname: Zou
authors.forename: Hong
authors.initials: H
authors.lastname: Yi
authors.forename: Mao-Yu
authors.initials: MY
authors.lastname: Li
authors.forename: Peng-Fei
authors.initials: PF
authors.lastname: Zhang
authors.forename: Cui
authors.initials: C
authors.lastname: Li
authors.forename: Fang
authors.initials: F
authors.lastname: Peng
authors.forename: Zhu-Chu
authors.initials: ZC
authors.lastname: Chen
authors.forename: Kai-Tai
authors.initials: KT
authors.lastname: Yao
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Xiao
mesh_terms: D016564:Amyloid beta-Protein Precursor / Q000276:immunology / Q000378:metabolism*; D000730:Androstadienes / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D055316:Cystatin C / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D009303:Nasopharyngeal Neoplasms / Q000378:metabolism / Q000473:pathology; D040901:Proteomics / Q000379:methods*; D011799:Quinazolines / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016211:Transforming Growth Factor alpha / Q000494:pharmacology; D000077191:Wortmannin
chemical_list: D016564:Amyloid beta-Protein Precursor; D000730:Androstadienes; D000911:Antibodies, Monoclonal; D055316:Cystatin C; D004791:Enzyme Inhibitors; D011799:Quinazolines; D016211:Transforming Growth Factor alpha; D066246:ErbB Receptors; C088860:4-((3-bromophenyl)amino)-6,7-dimethoxyquinazoline; D000077191:Wortmannin