Zhu-Chu Chen

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 Proteomic Analysis of Formalin-fixed and Paraffin-embedded Nasopharyngeal Carcinoma Using iTRAQ Labeling, Two-dimensional Liquid Chromatography, and Tandem Mass Spectrometry

Formalin-fixed, paraffin-embedded (FFPE) tissue specimens represent a potentially valuable resource for protein biomarker investigations. In this study, proteins were extracted by a heat-induced antigen retrieval technique combined with a retrieval solution containing 2% SDS from FFPE tissues of normal nasopharyngeal epithelial tissues (NNET) and three histological types of nasopharyngeal carcinoma (NPC) with diverse differentiation degrees. Then two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins among the types of NPC FFPE tissues. Our study resulted in the identification of 730 unique proteins, the distributions of subcellular localizations and molecular functions of which were similar to those of the proteomic database of human NPC and NNET that we had set up based on the frozen tissues. Additionally, the relative expression levels of cathepsin D, keratin8, SFN, and stathmin1 identified and quantified in this report were consistent with the immunohistochemistry results acquired in our previous study. In conclusion, we have developed an effective approach to identifying protein changes in FFPE NPC tissues utilizing iTRAQ technology in conjunction with an economical and easily accessible sample preparation method.

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.

Triosephosphate isomerase and peroxiredoxin 6, two novel serum markers for human lung squamous cell carcinoma

There is currently substantial interest in the identification of human tumor antigens for the diagnosis and immunotherapy of cancer. In our previous study, secretion character and up‐regulation of triosephosphate isomerase were observed in lung squamous cell carcinoma, and autoantibodies against triosephosphate isomerase and peroxiredoxin 6 were detected in the sera from over 25% of patients, but in none of the healthy controls. In this study, peroxiredoxin 6 was also found at higher levels in the sera of the patients. Up‐regulated triosephosphate isomerase and peroxiredoxin 6 were further validated by enzyme‐linked immunosorbent assay in an additional 61 lung squamous cell carcinoma patients, 23 lung adenocarcinoma patients, 56 other types of carcinoma patients, 12 benign lung disease patients, and 59 healthy controls. We found that both triosephosphate isomerase and peroxiredoxin 6 were specifically elevated in lung squamous cell carcinoma sera compared with other groups, with the exception of peroxiredoxin 6 in lung adenocarcinoma patients. Positive correlation between triosephosphate isomerase and distant metastasis was found. At the cut‐off point 0.221 (optical density value) on the receiver operating characteristic curve, triosephosphate isomerase could comparatively discriminate lung squamous cell carcinoma from healthy controls with a sensitivity of 65.6%, specificity 84.7%, and total accuracy 75%. For peroxiredoxin 6, at the cut‐off point 0.151, it could discriminate the two groups with a sensitivity of 70.5%, specificity 62.7%, and total accuracy 65.8%. With both triosephosphate isomerase and peroxiredoxin 6, discriminant analysis results showed that 68.9% of the lung squamous cell carcinoma and 83.1% of healthy controls were correctly classified. We concluded that triosephosphate isomerase and peroxiredoxin 6 could be markers for lung squamous cell carcinoma. (Cancer Sci 2009; 100: 2396–2401)

A subcelluar proteomic investigation into vincristine‐resistant gastric cancer cell line

Multidrug resistance (MDR) is a major obstacle to successful cancer treatment. To understand the mechanism of MDR better, a subcelluar proteomics approach was used to compare the protein profile between vincristine‐resistant human gastric cancer cell line SGC7901/VCR and its parental cell line SGC7901. After differential solubilization, the subfractionation proteins were separate by two‐dimensional gel electrophoresis (2‐DE), and the differential protein spots were identified by both MALDI‐TOF‐MS and ESI‐Q‐TOF‐MS. Then the differential expressional levels of partial identified proteins were determined by Western blot analysis. Furthermore, one of the highly expressed proteins in SGC7901/VCR, Sorcin, associated with MDR was analyzed. In this study, the well‐resolved, reproducible 2‐DE patterns of subfractionation proteins from SGC7901/VCR and SGC7901 were established, and 30 differential proteins between the two cell lines were identified. The functional validation showed that the elevated sorcin expression could contribute considerably to the vincristine resistance in SGC7901/VCR. The 30 differentially expressed proteins could be divided into six groups based on their functions: calcium binding proteins, chaperones, metabolic enzymes, proteins relative to signal transduction, proteins involved in transcription and translation, and transportation proteins, and most of them might be new MDR associated proteins, which have not been detected previously. These data will be valuable for further to study the mechanisms of MDR in human gastric cancer. J. Cell. Biochem. 104: 1010–1021, 2008.