Identification of the amyloid β-protein precursor and cystatin C as novel epidermal growth factor receptor regulated secretory proteins in nasopharyngeal carcinoma by proteomics.
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.
Tang
Can-E
CE
Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha 410008, China.
Guan
Yong-Jun
YJ
Yi
Bin
B
Li
Xin-Hui
XH
Liang
Ke
K
Zou
Hai-Yan
HY
Yi
Hong
H
Li
Mao-Yu
MY
Zhang
Peng-Fei
PF
Li
Cui
C
Peng
Fang
F
Chen
Zhu-Chu
ZC
Yao
Kai-Tai
KT
Xiao
Zhi-Qiang
ZQ
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
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