Jia-an Qu

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 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.

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.

MiR-23a sensitizes nasopharyngeal carcinoma to irradiation by targeting IL-8/Stat3 pathway

Radioresistance poses a major challenge in nasopharyngeal carcinoma (NPC) treatment, but little is known about how miRNA regulates this phenomenon. In this study, we investigated the function and mechanism of miR-23a in NPC radioresistance, one of downregulated miRNAs in the radioresistant NPC cells identified by our previous microarray analysis. We observed that miR-23a was frequently downregulated in the radioresistant NPC tissues, and its decrement correlated with NPC radioresistance and poor patient survival, and was an independent predictor for reduced patient survival. In vitro radioresponse assays showed that restoration of miR-23a expression markedly increased NPC cell radiosensitivity. In a mouse model, therapeutic administration of miR-23a agomir dramatically sensitized NPC xenografts to irradiation. Mechanistically, we found that reduced miR-23a promoted NPC cell radioresistance by activating IL-8/Stat3 signaling. Moreover, the levels of IL-8 and phospho-Stat3 were increased in the radioresistance NPC tissues, and negatively associated with miR-23a level. Our data demonstrate that miR-23a is a critical determinant of NPC radioresponse and prognostic predictor for NPC patients, and its decrement enhances NPC radioresistance through activating IL-8/Stat3 signaling, highlighting the therapeutic potential of miR-23a/IL-8/Stat3 signaling axis in NPC radiosensitization.

MiRNA-203 reduces nasopharyngeal carcinoma radioresistance by targeting IL8/AKT signaling

Radioresistance poses a major challenge in nasopharyngeal carcinoma (NPC) treatment, but little is known about how miRNA (miR) regulates this phenomenon. In this study, we investigated the function and mechanism of miR-203 in NPC radioresistance, one of downregulated miRs in the radioresistant NPC cells identified by our previous microarray analysis. We observed that miR-203 was frequently downregulated in the radioresistant NPC tissues compared with radiosensitive NPC tissues, and its decrement significantly correlated with NPC radioresistance and poor patient survival, and was an independent predictor for reduced patient survival. In vitro radioresponse assays showed that miR-203 mimic markedly decreased NPC cell radioresistance. In a mouse model, therapeutic administration of miR-203 agomir dramatically sensitized NPC xenografts to irradiation. Mechanistically, we confirmed that IL8 was a direct target of miR-203, and found that reduced miR-203 promoted NPC cell radioresistance by activating IL8/AKT signaling. Moreover, the levels of IL8 and phospho-AKT were significantly increased in the radioresistant NPC tissues compared with radiosensitive NPC tissues, and negatively associated with miR-203 level. Our data demonstrate that miR-203 is a critical determinant of NPC radioresponse, and its decrement enhances NPC radioresistance through targeting IL8/AKT signaling, highlighting the therapeutic potential of the miR-203/IL8/AKT signaling axis in NPC radiosensitization. Mol Cancer Ther; 14(11); 2653–64. ©2015 AACR.

Identification of flotillin-1 as a novel biomarker for lymph node metastasis and prognosis of lung adenocarcinoma by quantitative plasma membrane proteome analysis.

To identify a novel lung adenocarcinoma (AdC) biomarker, iTRAQ-tagging combined with 2D LC-MS/MS analysis was used to identify differentially expressed plasma membrane (PM) proteins in primary lung AdCs and paraneoplastic normal lung tissues (PNLTs). As a result, 36 differentially expressed membrane proteins were identified. Two differential PM proteins flotillin-1 and caveolin-1 were selectively validated by Western blotting. As there has been no report on the association of flotillin-1 with lung AdC, immunohistochemistry was further performed to detect the expression of flotillin-1 in the archival tissue specimens including 42 cases of PNLTs, 62 cases of primary lung AdCs with lymph node metastasis (LNM AdCs), and 46 cases of primary lung AdCs without lymph node metastasis (non-LNM AdCs), and the correlation of flotillin-1 expression levels in lung AdCs with clinicopathological features and clinical outcomes were evaluated. The results showed that up-regulation of flotillin-1 expression in lung AdCs was significantly correlated with advanced clinical stage, lymph node metastasis, increased postoperative relapse and decreased overall survival. Cox regression analysis revealed that the expressional level of flotillin-1 was an independent prognostic factor. The data suggest that flotillin-1 is a potential novel biomarker for lymph node metastasis and prognosis of lung AdC, and flotillin-1 up-regulation might play an important role in the pathogenesis of lung AdC.

The function and significance of SELENBP1 downregulation in human bronchial epithelial carcinogenic process.

Background Our quantitative proteomic study showed that selenium-binding protein 1 (SELENBP1) was progressively decreased in human bronchial epithelial carcinogenic process. However, there is little information on expression and function of SELENBP1 during human lung squamous cell cancer (LSCC) carcinogenesis. Methods iTRAQ-tagging combined with 2D LC-MS/MS analysis was used to identify differentially expressed proteins in the human bronchial epithelial carcinogenic process. SELENBP1, member of selenoproteins family and progressively downregulated in this process, was selected to further study. Both Western blotting and immunohistochemistry were performed to detect SELENBP1 expression in independent sets of tissues of bronchial epithelial carcinogenesis, and ability of SELENBP1 for discriminating NBE (normal bronchial epithelium) from preneoplastic lesions from invasive LSCC was evaluated. The effects of SELENBP1 downregulation on the susceptibility of benzo(a)pyrene (B[a]P)-induced human bronchial epithelial cell transformation were determined. Results 102 differentially expressed proteins were identified by quantitative proteomics, and SELENBP1 was found and confirmed being progressively decreased in the human bronchial epithelial carcinogenic process. The sensitivity and specificity of SELENBP1 were 80% and 79% in discriminating NBE from preneoplastic lesions, 79% and 82% in discriminating NBE from invasive LSCC, and 77% and 71% in discriminating preneoplastic lesions from invasive LSCC, respectively. Furthermore, knockdown of SELENBP1 in immortalized human bronchial epithelial cell line 16HBE cells significantly increased the efficiency of B[a]P-induced cell transformation. Conclusions The present data shows for the first time that decreased SELENBP1 is an early event in LSCC, increases B[a]P-induced human bronchial epithelial cell transformation, and might serve as a novel potential biomarker for early detection of LSCC.

Identification of the proteins related to p53-mediated radioresponse in nasopharyngeal carcinoma by proteomic analysis

Radiotherapy is the primary treatment for nasopharyngeal cancer (NPC), and p53 is closely associated with the radiosensitivity of cancer, but the molecular mechanisms of p53-mediated radioresponse in NPC remains unclear. We previously established NPC CNE2sip53 cell line with p53 knockdown and paired control cell line CNE2/pSUPER, which provides a cell model system to investigate mechanisms of p53-mediated radioresponse in NPC. In this study, we first compared the radiosensitivity of CNE2sip53 and CNE2/pSUPER by a clonogenic survival assay, cell growth assay, and Hoechst 33258 staining and flow cytometry analysis of apoptotic cells. The results showed that the radiosensitivity of CNE2sip53 was significantly lower than that of CNE2/pSUPER, indicating that p53 plays a role in mediating NPC radiosensitivity. To search for the proteins associated with the p53-mediated radioresponse in NPC, a proteomic approach was performed to identify the radioresponsive proteins in CNE2sip53 and CNE2p/SUPER, respectively, and then the difference of radioresponsive proteins in CNE2sip53 and CNE2p/SUPER was compared. As a result, 14 differential radioresponsive proteins were identified in the two cell lines, 4 proteins of which were conformed by Western blot. Among them, 9 and 5 proteins were identified solely from CNE2p/SUPER and CNE2sip53, respectively. Furthermore, protein-protein interaction analysis showed that 7 differential radioresponsive proteins identified only in CNE2p/SUPER were related to p53 protein. Our results suggest that the differential radioresponsive proteins unique to CNE2p/SUPER may be involved in p53-mediated radioresponse in NPC, which will be helpful for elucidating the mechanisms of p53-mediated NPC cellular response to radiotherapy.

Identification of heat shock protein 27 as a radioresistance-related protein in nasopharyngeal carcinoma cells.

PurposeTo identify the proteins involved in radioresistance in nasopharyngeal cancer (NPC) cells.MethodsSublethal ionizing radiation was applied to establish a radioresistant NPC cell line from its parental NPC cell line CNE1. Clonogenic survival assay, cell growth assay and flow cytometry analysis were used to examine the difference of radiosensitivity in the radioresistant CNE1 cells (CNE1-IR) and control CNE1 cells. Comparative proteomics was performed to identify the differential proteins in the two cell lines. Association of HSP27, one of upregulated proteins in CNE1-IR cells, with NPC cell radioresistance was selected for further investigation using antisense oligonucleotides (ASOs), clonogenic survival assay, Hoechst 33258 staining of apoptotic cells and MTT assay of cell viability.ResultsRadioresistant NPC cell line CNE1-IR derived from its parental cell line CNE1 was established. Thirteen differential proteins in the CNE1-IR and CNE1 cells were identified by proteomics, and differential expression of HSP27, one of identified proteins, was selectively confirmed by western blot. Inhibition of HSP27 expression by HSP27 ASOs decreased clonogenic survival and cell viability and increased cell apoptosis of CNE1-IR cells after irradiation, that is, enhanced radiosensitivity of CNE1-IR cells.ConclusionThe data suggest that HSP27 is a radioresistant protein in NPC cells, and its upregulation may be involved in the NPC radioresistance.

Identification of GLIPR1 tumor suppressor as methylation-silenced gene in acute myeloid leukemia by microarray analysis

PurposeTo identify methylation-silenced genes in acute myeloid leukemia (AML).MethodsMicroarray analyses were performed in AML cell line HL-60 cells exposed to the demethylating agent 5-aza-2dC. The methylation status and expression of glioma pathogenesis-related protein 1 (GLIPR1), one of highly induced genes by demethylation, were further detected in six hematopoietic malignancy cell lines and 260 bone marrow samples from leukemia patients and nonmalignant diseases as control, as well as pre-treated and post-treated bone marrow samples from 24 complete remission AML patients received chemotherapy using MS-PCR, bisulfite DNA sequencing, RT-PCR, and Western blotting.ResultsOne hundred and nine genes were significantly induced by demethylation in HL-60 cells, 12 genes of which were confirmed by RT-PCR. GLIPR1, a tumor suppressor gene, was frequently methylation-silenced in AML cell lines and AML patients, but not in the other hematopoietic malignancy cell lines and patients. The frequencies of methylation-silenced GLIPR1 in the pre-treatment were significantly higher than those in the post-treatment in complete remission AML patients.ConclusionWe identify 109 genes induced by demethylation in HL-60 cells, and demonstrate that GLIPR1 is a methylation-silenced gene in the AML patients, and may serve as a marker for monitoring disease activity during therapy in the AML patients. The data provide the important information for studying the pathogenesis of AML and discovering the target genes of methylating agents.