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Analysis of EGFR signaling pathway in nasopharyngeal carcinoma cells by quantitative phosphoproteomics.

ABSTRACT: BACKGROUND:The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, the downstream signaling proteins of EGFR in NPC have not yet been completely understood at the system level. The aim of this study was identify novel downstream proteins of EGFR signaling pathway in NPC cells. RESULTS:We analyzed EGFR-regulated phosphoproteome in NPC CNE2 cells using 2D-DIGE and mass spectrometry analysis after phosphoprotein enrichment. As a result, 33 nonredundant phosphoproteins including five known EGFR-regulated proteins and twenty-eight novel EGFR-regulated proteins in CNE2 were identified, three differential phosphoproteins were selectively validated, and two differential phosphoproteins (GSTP1 and GRB2) were showed interacted with phospho-EGFR. Bioinformatics analysis showed that 32 of 33 identified proteins contain phosphorylation modification sites, and 17 identified proteins are signaling proteins. GSTP1, one of the EGFR-regulated proteins, associated with chemoresistance was analyzed. The results showed that GSTP1 could contribute to paclitaxel resistance in EGF-stimulated CNE2 cells. Furthermore, an EGFR signaling network based on the identified EGFR-regulated phosphoproteins were constructed using Pathway Studio 5.0 software, which includes canonical and novel EGFR-regulated proteins and implicates the possible biological roles for those proteins. CONCLUSION:The data not only can extend our knowledge of canonical EGFR signaling, but also will be useful to understand the molecular mechanisms of EGFR in NPC pathogenesis and search therapeutic targets for NPC.

SUBMITTER: Ruan L

PROVIDER: S-EPMC3141626 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Analysis of EGFR signaling pathway in nasopharyngeal carcinoma cells by quantitative phosphoproteomics.

Ruan Lin L Li Xin-Hui XH Wan Xun-Xun XX Yi Hong H Li Cui C Li Mao-Yu MY Zhang Peng-Fei PF Zeng Gu-Qing GQ Qu Jia-Quan JQ He Qiu-Yan QY Li Jian-Huang JH Chen Yu Y Chen Zhu-Chu ZC Xiao Zhi-Qiang ZQ

Proteome science 20110628

<h4>Background</h4>The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, the downstream signaling proteins of EGFR in NPC have not yet been completely understood at the system level. The aim of this study was identify novel downstream proteins of EGFR signaling pathway in NPC cells.<h4>Results</h4>We analyzed EGFR-regulated phosphoproteome in NPC CNE2 cells using 2D-DIGE and mass spectrometry an ...[more]

PMID: 21711528

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Project description:BACKGROUND:Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic cancer. N,N'-dinitrosopiperazine (DNP), a carcinogen with specificity for nasopharyngeal epithelium, facilitates NPC metastasis. However, the underlying mechanism is not known. METHODS:Quantitative phosphoproteomics, using stable isotope labeling of amino acids in cell cultures, was employed to identify phosphoproteins associated with NPC metastasis mediated by DNP. NPC cell line 6-10B, which is relatively less metastatic, was used to investigate DNP-mediated metastasis. Boyden chamber invasion assay was used to measure DNP-induced motility and invasion, and nude mice were used to verify DNP-mediated metastasis in vivo. Several different phosphoproteins detected by proteomics analysis were verified by immunoblotting. DNP-mediated metastasis facilitated by lysine-rich CEACAM1 co-isolated protein (LYRIC) phosphorylation at serine 568 was confirmed using mutations targeting the phosphorylation site of LYRIC. DNP-mediated metastasis through LYRIC phosphorylation was confirmed in the NPC cell line CNE1. DNP-mediated LYRIC phosphorylation at serine 568 was also verified in metastatic tumors of BABL/c nude mice. RESULTS:Boyden chamber invasion assay indicated that DNP mediated cell motility and invasion of NPC cell 6-10B in vitro, and experiments with nude mice indicated that DNP increased 6-10B metastasis in vivo. In the phosphoproteomics analysis, we detected 216 phosphorylation sites on 130 proteins; among these, 48 phosphorylation sites on 30 unique phosphopeptides were modulated by DNP by at least 1.5-fold. DNP mediated the expression of phosphorylated GTPase, ferritin, LYRIC, and RNA polymerase, and it decreased the expression of phosphorylated torsin-1A protein 1. Furthermore, DNP induced LYRIC phosphorylation at serine 568 to facilitate cell motility and invasion, whereas DNP-mediated motility and invasion was decreased when serine 568 in LYRIC was mutated. In another NPC cell line, CNE1, DNP also mediated cell motility and invasion followed by enhanced phosphorylation of LYRIC at serine 568. Finally, phosphorylated-LYRIC expression at serine 568 was significantly increased in metastatic tumors induced by DNP. CONCLUSION:DNP regulates multiple signaling pathways through protein phosphorylation, including the phosphorylation of LYRIC at serine 568, and mediates NPC metastasis. These findings provide insights on the complexity and dynamics of DNP-facilitated metastasis, and may help to gain a better understanding of the mechanisms by clarifying NPC-induced metastasis.

Dataset Information

Analysis of EGFR signaling pathway in nasopharyngeal carcinoma cells by quantitative phosphoproteomics.

Publications

Analysis of EGFR signaling pathway in nasopharyngeal carcinoma cells by quantitative phosphoproteomics.

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