Zhi-Qiang Xiao

Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin.

EGFR is a potent stimulator of invasion and metastasis in head and neck squamous cell carcinomas (HNSCC). However, the mechanism by which EGFR may stimulate tumor cell invasion and metastasis still need to be elucidated. In this study, we showed that activation of EGFR by EGF in HNSCC cell line SCC10A enhanced cell migration and invasion, and induced loss of epitheloid phenotype in parallel with downregulation of E‐cadherin and upregulation of N‐cadherin and vimentin, indicating that EGFR promoted SCC10A cell migration and invasion possibly by an epithelial to mesenchymal transition (EMT)‐like phenotype change. Interestingly, activation of EGFR by EGF induced production of matrix metalloproteinase‐9 (MMP‐9) and soluble E‐cadherin (sE‐cad), and knockdown of MMP‐9 by siRNA inhibited sE‐cad production induced by EGF in SCC10A. Moreover, both MMP‐9 knockdown and E‐cadherin overexpression inhibited cell migration and invasion induced by EGF in SCC10A. The results indicate that EGFR activation promoted cell migration and invasion through inducing MMP‐9‐mediated degradation of E‐cadherin into sE‐cad. Pharmacologic inhibition of EGFR, MEK, and PI3K kinase activity in SCC10A reduced phosphorylated levels of ERK‐1/2 and AKT, production of MMP‐9 and sE‐cad, cell migration and invasion, and expressional changes of EMT markers (E‐cadherin and N‐cadherin) induced by EGF, indicating that EGFR activation promotes cell migration and invasion via ERK‐1/2 and PI3K‐regulated MMP‐9/E‐cadherin signaling pathways. Taken together, the data suggest that EGFR activation promotes HNSCC SCC10A cell migration and invasion by inducing EMT‐like phenotype change and MMP‐9‐mediated degradation of E‐cadherin into sE‐cad related to activation of ERK‐1/2 and PI3K signaling pathways. J. Cell. Biochem. 112: 2508–2517, 2011.

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

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.

Identification of Candidate Biomarkers for Early Detection of Human Lung Squamous Cell Cancer by Quantitative Proteomics

To discover novel biomarkers for early detection of human lung squamous cell cancer (LSCC) and explore possible mechanisms of LSCC carcinogenesis, iTRAQ-tagging combined with two dimensional liquid chromatography tandem MS analysis was used to identify differentially expressed proteins in human bronchial epithelial carcinogenic process using laser capture microdissection-purified normal bronchial epithelium (NBE), squamous metaplasia (SM), atypical hyperplasia (AH), carcinoma in situ (CIS) and invasive LSCC. As a result, 102 differentially expressed proteins were identified, and three differential proteins (GSTP1, HSPB1 and CKB) showing progressively expressional changes in the carcinogenic process were selectively validated by Western blotting. Immunohistochemistry was performed to detect the expression of the three proteins in an independent set of paraffin-embedded archival specimens including various stage tissues of bronchial epithelial carcinogenesis, and their ability for early detection of LSCC was evaluated by receiver operating characteristic analysis. The results showed that the combination of the three proteins could perfectly discriminate NBE from preneoplastic lesions (SM, AH and CIS) from invasive LSCC, achieving a sensitivity of 96% and a specificity of 92% in discriminating NBE from preneoplatic lesions, a sensitivity of 100% and a specificity of 98% in discriminating NBE from invasive LSCC, and a sensitivity of 92% and a specificity of 91% in discriminating preneoplatic lesions from invasive LSCC, respectively. Furthermore, we knocked down GSTP1 in immortalized human bronchial epithelial cell line 16HBE cells, and then measured their susceptibility to carcinogen benzo(a)pyrene-induced cell transformation. The results showed that GSTP1 knockdown significantly increased the efficiency of benzo(a)pyrene-induced 16HBE cell transformation. The present data first time show that GSTP1, HSPB1 and CKB are novel potential biomarkers for early detection of LSCC, and GSTP1 down-regulation is involved in human bronchial epithelial carcinogenesis.