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Dinitrosopiperazine-mediated phosphorylated-proteins are involved in nasopharyngeal carcinoma metastasis.

ABSTRACT: N,N'-dinitrosopiperazine (DNP) with organ specificity for nasopharyngeal epithelium, is involved in nasopharyngeal carcinoma (NPC) metastasis, though its mechanism is unclear. To reveal the pathogenesis of DNP-induced metastasis, immunoprecipitation was used to identify DNP-mediated phosphoproteins. DNP-mediated NPC cell line (6-10B) motility and invasion was confirmed. Twenty-six phosphoproteins were increased at least 1.5-fold following DNP exposure. Changes in the expression levels of selected phosphoproteins were verified by Western-blotting analysis. DNP treatment altered the phosphorylation of ezrin (threonine 567), vimentin (serine 55), stathmin (serine 25) and STAT3 (serine 727). Furthermore, it was shown that DNP-dependent metastasis is mediated in part through ezrin at threonine 567, as DNP-mediated metastasis was decreased when threonine 567 of ezrin was mutated. Strikingly, NPC metastatic tumors exhibited a higher expression of phosphorylated-ezrin at threonine 567 than the primary tumors. These findings provide novel insight into DNP-induced NPC metastasis and may contribute to a better understanding of the metastatic mechanisms of NPC tumors.

SUBMITTER: Tan G

PROVIDER: S-EPMC4264155 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Dinitrosopiperazine-mediated phosphorylated-proteins are involved in nasopharyngeal carcinoma metastasis.

Tan Gongjun G Tang Xiaowei X Huang Damao D Li Yuejin Y Liu Na N Peng Zhengke Z Zhang Zhenlin Z Duan Chaojun C Lu Jinping J Yan Guangrong G Tang Faqing F

International journal of molecular sciences 20141104 11

N,N'-dinitrosopiperazine (DNP) with organ specificity for nasopharyngeal epithelium, is involved in nasopharyngeal carcinoma (NPC) metastasis, though its mechanism is unclear. To reveal the pathogenesis of DNP-induced metastasis, immunoprecipitation was used to identify DNP-mediated phosphoproteins. DNP-mediated NPC cell line (6-10B) motility and invasion was confirmed. Twenty-six phosphoproteins were increased at least 1.5-fold following DNP exposure. Changes in the expression levels of selecte ...[more]

PMID: 25375189

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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.

Project description:BACKGROUND: Mitochondrial proteomic alterations of nasopharyngeal carcinoma metastasis remain unknown. Our purpose is to screen mitochondrial proteins for the elucidation of the molecular mechanisms of nasopharyngeal carcinoma metastasis and the discovery of metastasis-related biomarkers. METHODS: Mitochondria were isolated from nasopharyngeal carcinoma metastatic (5-8F) and nonmetastatic (6-10B) cell lines, respectively. After characterization of isolated mitochondria, mitochondrial differentially expressed proteins (DEPs) were quantified by two-dimensional difference in-gel electrophoresis (2D-DIGE), and identified by peptide mass fingerprint (PMF) and tandem mass spectrometry (MS/MS). A functional enrichment analysis and a protein-protein interaction sub-network analysis for DEPs were carried out with bioinformatics. Furthermore, siRNAs transient transfections were used to suppress expressions of some up-regulated DEPs in metastatic cells (5-8F), followed by Transwell Migration assay. RESULTS: Sixteen mitochondrial DEPs including PRDX3 and SOD2 were identified. Those 5-8F cells with suppression of PRDX3 showed an increased mobility potential. The functional enrichment analyses of DEPs discovered five significant biological processes including cellular response to reactive oxygen species, hydrogen peroxide metabolic process, regulation of mitochondrial membrane potential, cell redox homeostasis and oxidation reduction, and five significant molecular functions including oxidoreductase activity, caspase inhibitor activity, peroxiredoxin activity, porin activity and antioxidant activity. A protein-protein interaction sub-network of DEPs was generated with literature data. Ten mitochondrial DEPs including PRDX3, PRDX6, SOD2, ECH1, SERPINB5, COX5A, PDIA5, EIF5A, IDH3B, and PSMC4 were rationalized in the tumor-stroma co-evolution model that mitochondrial oxidative stress directly contributes to tumor metastasis. CONCLUSIONS: Sixteen mitochondrial DEPs were identified with mass spectrometry and ten of them were rationalized in the tumor-stroma co-evolution model. Those 5-8F cells with suppression of PRDX3 showed an increased mobility potential. These data suggest that those mitochondrial DEPs are potential biomarkers for NPC metastasis, and their dysregulation would play important roles in mitochondria oxidative stress-mediated NPC metastatic process.

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Dinitrosopiperazine-mediated phosphorylated-proteins are involved in nasopharyngeal carcinoma metastasis.

Publications

Dinitrosopiperazine-mediated phosphorylated-proteins are involved in nasopharyngeal carcinoma metastasis.

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