Can-E Tang

Identification of Biomarkers for Predicting Nasopharyngeal Carcinoma Response to Radiotherapy by Proteomics

Radiotherapy is the primary treatment for nasopharyngeal cancer (NPC), but radioresistance remains a serious obstacle to successful treatment in many cases. To identify the proteins involved in this resistance and to evaluate their potential for predicting NPC response to radiotherapy, we first established a radioresistant subclone cell line (CNE2-IR) derived from NPC cell line CNE2 by treating the cells with five rounds of sublethal ionizing radiation. Proteomics was then performed to compare the protein profiles of CNE2-IR and CNE2, and a total of 34 differential proteins were identified. Among them, 14-3-3sigma and Maspin were downregulated and GRP78 and Mn-SOD were upregulated in the radioresistant CNE2-IR compared with control CNE2, which was conformed by Western blot. Immunohistochemistry was performed to detect the expression of the four validated proteins in the 39 radioresistant and 51 radiosensitive NPC tissues and their value for predicting NPC response to radiotherapy were evaluated by receiver operating characteristic analysis. The results showed that the downregulation of 14-3-3sigma and Maspin and the upregulation of GRP78 and Mn-SOD were significantly correlated with NPC radioresistance and the combination of the four proteins achieved a sensitivity of 90% and a specificity of 88% in discriminating radiosensitive from radiaoresistant NPC. Furthermore, the resistance to ionizing radiation can be partially reversed by the overexpression of 14-3-3sigma in the CNE2-IR. The data suggest that 14-3-3sigma, Maspin, GRP78, and Mn-SOD are potential biomarkers for predicting NPC response to radiotherapy and their dysregulation may be involved in the radioresistance of NPC.

Inactivation of 14‐3‐3 σ by promoter methylation correlates with metastasis in nasopharyngeal carcinoma

14‐3‐3 σ, the downstream target of p53, is a negative regulator of cell cycle G2‐M phase checkpoint in response to DNA damage. Our previous comparative proteomics study showed that 14‐3‐3 σ was downregulated or lost in nasopharyngeal carcinoma (NPC) tissue compared with non‐cancerous nasopharyngeal epithelial tissue (NNET). In this study, we further investigated for the epigenetic mechanism of 14‐3‐3 σ inactivation. Methylation‐specific PCR showed 14‐3‐3 σ promoter methylation in 100% of analyzed NPC cell lines (4/4) but not in immortalized human nasopharyngeal epithelial cell line NP69. Treatment of the four NPC cell lines with the methyltransferase inhibitor 5‐aza‐2′‐dC resulted in the demethylation and upregulation of 14‐3‐3 σ. In tissues, 14‐3‐3 σ promoter methylation occurred at a higher frequency in NPC, 63/75 (84%), compared to adjacent NNET, 7/25 (28%), and fully methylated 14‐3‐3 σ promoter was detected in NPC but not in any of adjacent NNET. RT‐PCR, Western blotting, and immunohistochemistry showed that 14‐3‐3 σ expression was downregulated or lost in NPC with methylation, and there was a negative correlation between the expression levels and methylation statuses of 14‐3‐3 σ gene. In addition, the patients with methylated 14‐3‐3 σ presented a higher frequency of lymph node and distant metastasis, and an advanced clinical stage, and overexpression of 14‐3‐3 σ in NPC cell line 5‐8F with high metastatic potential was able to inhibit its in vitro invasive ability. Our data are the first to show that 14‐3‐3 σ is frequently inactivated by promoter methylation in NPC and this aberrant methylation correlates with lymph node and distant metastasis. J. Cell. Biochem. 106: 858–866, 2009.

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.

Identification of RKIP as a differentially tyrosine-phosphorylated protein in nasopharyngeal carcinoma and normal nasopharyngeal epithelial tissues by phosphoproteomic approach

Aims To screen for differentially tyrosine-phosphorylated proteins between nasopharyngeal carcinoma (NPC) and normal nasopharyngeal epithelial tissues (NNET) to provide a basis for elucidate the molecular mechanisms of NPC carcinogenesis. Methods Two-dimensional (2-D) electrophoresis was applied to separate proteins from NPC and NNET, respectively, and 2-D Western blotting was performed to detect tyrosine-phosphorylated proteins using antiphosphotyrosine antibody. Differentially tyrosine-phosphorylated proteins were identified by electrospray ionization-quadrupole time-of-flight MS (ESI-Q-TOF MS). NetPhos software was used to predict the tyrosine-phosphorylation sites of the identified proteins, and Western blotting was used to detect the tyrosine-phosphorylated levels of RKIP in NPC and NNET. Results Twenty-five differentially tyrosine-phosphorylated proteins in the two types of tissues were found, 13 of which were identified by ESI-Q-TOF MS. Among the 13 identified proteins, tyrosine-phosphorylated levels of 7 proteins were increased, and those of 6 proteins were decreased in NPC compared with NNET. NetPhos software prediction showed that all the 13 identified proteins contained tyrosine phosphorylation sites, and the differentially tyrosine-phosphorylated level of RKIP in NPC and NNET was confirmed. Conclusion The 13 differentially tyrosine-phosphorylated proteins may be involved in the development and progression of NPC.

Identification of tyrosine phosphoproteins in signaling pathway triggered TGF-a by using functional proteomics technology

Although several groups had conducted proteomics of nasopharyngeal carcinoma (NPC), little study was involved in phosphoproteomics, oncogenic signaling, and cancer research about NPC. Analysis of phosphotyrosine proteins from transforming growth factor alpha (TGF-a) triggered phosphotyrosine proteome permitted the identification of novel downstream substrates of the epidermal growth factor receptor (EGFR). Using functional proteomics technology based on 2-DE, 2-D western blotting, and mass spectrometry, we identified and quantified the tyrosine phosphorylation levels of 16 proteins between control and TGF-a-treated CNE2 human NPC cells. Among these proteins, tyrosine phosphorylated levels of ten proteins were increased, and those of six proteins were decreased in TGF-a-treated CNE2 cells compared with control. In addition, among these identified proteins, ANXA3, KRT8, and KRT18 were validated to be novel tyrosine-phosphorylation targets of EGFR signaling by IP-western blotting and part of a complex EGFR phosphotyrosine signaling network. These novel findings will provide new insights into the complex EGFR phosphorylation signaling and may have implications in molecular cancer therapy of NPC.

Identification of integrin β1 as a prognostic biomarker for human lung adenocarcinoma using 2D-LC-MS/MS combined with iTRAQ technology

To discover novel lung adenocarcinoma (AdC) biomarkers, isobaric tags for relative and absolute quantitation (iTRAQ)-tagging combined with 2D-LC-MS/MS analysis was used to identify differentially expressed plasma membrane proteins in lung AdC and paired paraneoplastic normal lung tissues (PNLTs) adjacent to tumors. In this study, significant caveolin-1 downregulation and integrin β1 upregulation was observed in primary lung AdC vs. PNLT. As there has been no report on the association of integrin β1 with lung AdC, immunohistochemical staining was performed to detect the expression of integrin β1 in an independent set of archival tissue specimens including 42 cases of PLNT, 46 cases of without lymph node metastasis primary AdC (non-LNM AdC) and 62 cases of LNM AdC; the correlation of their expression levels with clinicopathological characteristics and clinical outcomes were evaluated. Based on the data, upregulation of integrin β1 was significantly correlated with advanced clinical stage and lymph node metastasis. Integrin β1 overexpression was significantly associated with advanced clinical stage (P<0.05), lymph node metastasis (P<0.05), increased relapse rate (P<0.05) and decreased overall survival (P<0.05) in AdCs. Cox regression analysis indicated that integrin β1 overexpression is an independent prognostic factor. The data suggest that integrin β1 is a potential biomarker for LNM and prognosis of AdC and integrin β1 upregulation may play an important role in the pathogenesis of AdC.

Association of BTBD7 with Metastasis and Poor Prognosis in Non-Small-Cell Lung Cancer Patients

Metastasis in lung cancer portends a poor prognosis, and the epithelial-mesenchymal transition (EMT) in lung cancer cells is considered a prerequisite to achieve metastatic potential. Recent studies indicate that BTB/POZ domain-containing protein 7 (BTBD7) regulates EMT-associated proteins in human malignancies and however, the role of BTBD7 in lung cancer have not been identified. In present study, we examined BTBD7 expression status and its association with unfavorable clinical features in non-small-cell lung cancer (NSCLC). Firstly, we studied the fresh specimens, and found that both mRNA and protein expression levels of BTBD7 in NSCLC tissue were significantly increased compared with the adjacent nontumorous lung tissue. Then, we determined BTBD7 protein expressions in the paraffin-embedded samples from NSCLC patients, and analyzed the relations of BTBD7 expression with clinicopathologic features of the patients. The results showed that incidence of metastasis in patients with positive BTBD7 expression was significantly higher than that in those with negative BTBD7 expression, and the positive BTBD7 expression rate in metastatic cases was significantly higher than that in non-metastatic ones; furthermore, Cox regression analyses revealed that BTBD7 was an independent risk factor for either metastasis or survival in NSCLC patients. Thus, we conclude that BTBD7 contributes to metastasis of NSCLC and BTBD7-positive NSCLC may have a high potential for metastasis and thereby a poor prognosis

Enhancing the stability of 18O-labeled peptides through removal of immobilized trypsin by ZipTips

Trypsin-catalyzed ¹⁸O labeling is increasingly used in shotgun proteomics for relative peptide/protein quantitation. However, precise quantitative measurements are often complicated by the instability of ¹⁸O-labeled peptides caused mainly by oxygen back-exchange. Although a number of attempts have been made to reduce or prevent oxygen back-exchange, there is still room for improvement. Here we demonstrate that the removal of immobilized trypsin by filtration using ZipTips can efficiently minimize oxygen back-exchange and enhance the stability of ¹⁸O-labeled peptides under various pH conditions. The ¹⁸O-labeled peptides processed by the approach were successfully separated by immobilized pH gradient-isoelectric focusing (IPG-IEF), and no marked decrease in the extent of labeling was observed. The results also demonstrated that there was no correlation between the extent of ¹⁸O labeling and molecular weight or isoelectric point (pI). The approach presented here is especially applicable to microscale samples. Its ability to generate stably ¹⁸O-labeled samples without back-exchange should expand the application scope of the ¹⁸O-labeling technique.