Peng-Fei Zhang

Identification of Novel Nasopharyngeal Carcinoma Biomarkers by Laser Capture Microdissection and Proteomic Analysis

Purpose: To identify novel nasopharyngeal carcinoma (NPC) biomarkers by laser capture microdissection and a proteomic approach. Experimental Design: Proteins from pooled microdissected NPC and normal nasopharyngeal epithelial tissues (NNET) were separated by two-dimensional gel electrophoresis, and differential proteins were identified by mass spectrometry. Expression of three differential proteins (stathmin, 14-3-3σ, and annexin I) in the above two tissues as well as four NPC cell lines was determined by Western blotting. Immunohistochemistry was also done to detect the expression of three differential proteins in 98 cases of primary NPC, 30 cases of NNET, and 20 cases of cervical lymph node metastases, and the correlation of their expression levels with clinicopathologic features and clinical outcomes were evaluated. Results: Thirty-six differential proteins between the NPC and NNET were identified. The expression levels of stathmin, 14-3-3σ, and annexin I in the two types of tissues were confirmed and related to differentiation degree and/or metastatic potential of the NPC cell lines. Significant stathmin up-regulation and down-regulation of 14-3-3σ and annexin I were observed in NPC versus NNET, and significant down-regulation of 14-3-3σ and annexin I was also observed in lymph node metastasis versus primary NPC. In addition, stathmin up-regulation and down-regulation of 14-3-3σ and annexin I were significantly correlated with poor histologic differentiation, advanced clinical stage, and recurrence, whereas down-regulation of 14-3-3σ and annexin I was also significantly correlated with lymph node and distant metastasis. Furthermore, survival curves showed that patients with stathmin up-regulation and down-regulation of 14-3-3σ and annexin I had a poor prognosis. Multivariate analysis revealed that the expression status of stathmin, 14-3-3σ, and annexin I was an independent prognostic indicator. Conclusion: The data suggest that stathmin, 14-3-3σ, and annexin I are potential biomarkers for the differentiation and prognosis of NPC, and their dysregulation might play an important role in the pathogenesis of NPC.

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.

Identification of metastasis associated proteins in human lung squamous carcinoma using two-dimensional difference gel electrophoresis and laser capture microdissection

A quantitative proteomic approach was used to discover potential protein markers associated with lymph node metastasis (LNM) in human lung squamous carcinoma (LSC). Laser capture microdissection was performed to purify LSC cells with LNM (LNM LSC) and LSC without LNM (non-LNM LSC). The differentially expressed proteins between pooled microdissected non-LNM LSC and LNM LSC cells were identified by two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry (MS). 14 proteins were found to be differentially expressed between non-LNM LSC and LNM LSC. Among these proteins, ten proteins were overexpressed in LNM LSC compared with non-LNM LSC, and four proteins were downregulated in LNM LSC. Some of these identified proteins (Annexin A2, HSP27, CK19, and 14-3-3sigma) were further confirmed by Western blotting and immunohistochemical analysis. These results show the value of LCM coupled with 2D-DIGE in identifying potential markers for lymph node metastasis of LSC, and also provide further insights into the prognosis of LSC.

Quantitative proteome analysis reveals annexin A3 as a novel biomarker in lung adenocarcinoma.

Metastasis is a common phenomenon and the major lethal cause of lung adenocarcinoma (AdC). To discover novel potential biomarkers associated with lymph node metastasis and prognosis in lung AdC, we assessed differences in protein expression between primary lung AdC with (LNM AdC) and without lymph node metastasis (non‐LNM AdC) using a quantitative proteomic approach. Laser capture microdissection was performed to purify the cancer cells from primary lung AdC tissues. The differential proteins between the pooled microdissected non‐LNM AdC and LNM AdC tissues were identified by two‐dimensional difference gel electrophoresis (2D‐DIGE) coupled with mass spectrometry (MS). In this study, twenty proteins were found to be differentially expressed in two types of lung AdC. ANXA3, significantly up‐regulated in LNM AdC compared with non‐LNM AdC, was validated by western blotting. Immunohistochemistry showed that ANXA3 over‐expression was frequently observed in LNM AdCs and matched lymph node metastases compared with non‐LNM AdCs. ANXA3 over‐expression was significantly associated with advanced clinical stage (p < 0.001) and lymph node metastasis (p < 0.001) and increased relapse rate (p < 0.001) and decreased overall survival (p < 0.001) in lung AdCs. Cox regression analysis indicated ANXA3 over‐expression was an independent prognostic factor. Our results indicate that ANXA3 might serve as a novel biomarker for lymph node metastasis and prognosis in lung AdC, and play an important role in lung AdC progression. Copyright

Identificating Cathepsin D as a Biomarker for Differentiation and Prognosis of Nasopharyngeal Carcinoma by Laser Capture Microdissection and Proteomic Analysis

In this study, we applied laser capture microdissection and a proteomic approach to identify novel nasopharyngeal carcinoma (NPC) biomarkers. Proteins from pooled microdissected NPC and normal nasopharyngeal epithelial tissues (NNET) were separated by two-dimensional gel electrophoresis, and differential proteins were identified by mass spectrometry. Expression of the differential protein cathepsin D in the above two tissues as well as four NPC cell lines was determined by Western blotting. Next, siRNA was used to inhibit the expression of cathepsin D in highly metastatic NPC cell line 5-8F to examine whether it associates with NPC metastasis. Immunohistochemistry was also performed to detect the expression of cathepsin D in 72 cases of primary NPC, 28 cases of NNET, and 20 cases of cervical lymph node metastases, and the correlation of its expression level with clinicopathologic features and clinical outcomes were evaluated. Thirty-six differential proteins between the NPC and NNET were identified. The expression level of cathepsin D in the two types of tissues was confirmed by Western blotting and related to differentiation degree and metastatic potential of the NPC cell lines. Down-regulated cathepsin D expression by siRNA significantly decreased in vitro invasive ability of 5-8F cells. Significant cathepsin D down-regulation was observed in NPC versus NNET, whereas significant cathepsin D up-regulation was observed in lymph node metastasis versus primary NPC. In addition, cathepsin D down-regulation was significantly correlated with poor histological differentiation, whereas cathepsin D up-regulation was significantly correlated with advanced clinical stage, recurrence, and lymph node and distant metastasis. Furthermore, survival curves showed that patients with cathepsin D up-regulation had a poor prognosis. Multivariate analysis confirmed that cathepsin D expression was an independent prognostic indicator. The data suggest that cathepsin D is a potential biomarker for the differentiation and prognosis of NPC, and its dysregulation might play an important role in the pathogenesis of NPC.

Identification of RKIP as an invasion suppressor protein in nasopharyngeal carcinoma by proteomic analysis.

To identify novel proteins associated with the pathogenesis of nasopharyngeal carcinoma (NPC), a proteomic approach was used to screen for differential proteins between NPC and adjacent noncancerous nasopharyngeal epithelial tissue (ANNET). As a result, 21 differential proteins were identified by two-dimensional electrophoresis and mass spectrometer. Raf kinase inhibitor protein (RKIP), one of the downregulated proteins in NPC compared to ANNET, was investigated for its role in the metastasis of NPC. Western blot analysis and immunohistochemistry were used to detect RKIP expression in 5-8F and 6-10B NPC cell lines with the different metastatic potentials, and in NNET, primary NPC and NPC metastasis. Furthermore, high metastatic 5-8F with low RKIP expression and nonmetastatic 6-10B with high RKIP expression were stably transfected with plasmids that expressed sense and antisense RKIP cDNA, respectively, or with empty vector. The effects of RKIP expression on in vitro cell invasion, and the activity of Raf-1/MEK/ERK signaling pathway were analyzed in the transfected cells. The results showed that RKIP was significantly downregulated in 5-8F compared with 6-10B, in NPC compared with NNET, and not detectable in NPC metastasis. Overexpressed RKIP in 5-8F could decrease its in vitro cell invasion, whereas downregulated RKIP in 6-10B could increase its in vitro cell invasion. RKIP negatively regulated Raf-1/MEK/ERK signaling pathway in NPC cells, and activation of this signaling pathway by RKIP downregulation increased in vitro invasion of NPC cells. Taken together, our results suggest that RKIP may be a NPC metastasis suppressor, and decreased RKIP expression is associated with the increased invasive capability of NPC cells possibly through the activation of Raf-1/MEK/ERK pathway.

Identification of differential proteins in nasopharyngeal carcinoma cells with p53 silence by proteome analysis

Although mutation of p53 tumor‐suppressor gene is rare in nasopharyngeal carcinoma (NPC), NPC has a high frequency of overexpression of p53 protein. There seem to be complex mechanisms of inactivation and stabilization of p53 in NPC. To detect proteins associated with the function of p53 in high throughout screening, we succeeded in establishing p53 knockdown human NPC CNE2 cell line (CNE2sip53) using stable RNA interference, and compared the proteomic changes between CNE2sip53 and control cell line CNE2/pSUPER using two‐dimensional gel electrophoresis. Twenty‐two differentially expressed proteins between the two cell lines were identified by both matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry and electrospray ionization tandem mass spectrometry, some of which are known to be associated with the p53 function (HSP27, hnRNP K, 14‐3‐3σ, etc.), and others may be novel proteins associated with p53 function (eIF4B, TPT1, hnRNP H3, SFRS1 etc.). Furthermore, several differential proteins including HSP27, HSP70, GRP75 and GRP78 were verified as p53 interacting proteins in NPC by immunoprecipitation and Western blot analysis, and the suppression of HSP27 expression by HSP27 antisense oligonucleotides could decrease the p53 protein level. Our data suggest that these differential proteins may be associated with the function of p53 in NPC, and provide new clues to elucidate the mechanisms of inactivation and stabilization of p53 in 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.

Identificating 14-3-3 sigma as a lymph node metastasis-related protein in human lung squamous carcinoma.

In this study, we aim to screen metastasis-related proteins in human lung squamous carcinoma (LSC) using laser capture microdissection and a proteomic approach. Twenty two differential proteins were identified from pooled microdissected primary LSC and matched lymph node (LN) metastatic tissues. Expression of the differential protein 14-3-3 sigma was determined by Western blotting and immunohistochemistry. In cell invasion assay, down-regulated 14-3-3 sigma by siRNA increased in vitro invasive ability of HTB-182 and A549 cells, up-regulation of 14-3-3 sigma by pcDNA3.0/14-3-3 sigma decreased in vitro invasive ability of HTB-182 and A549 cells. The data suggest that 14-3-3 sigma is a potential LN metastasis-related protein in LSC, and its dysregulation might play an important role in the LN metastatic process of LSC.

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

BackgroundThe 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.ResultsWe 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.ConclusionThe 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.