| Nasopharyngeal carcinoma(NPC) is one of the most frequently diagnosed malignancies of head and neck with high incidence in Southern China, especially in Guangdong, Guangxi, Hunan, Fujian and Jiangxi province. Because of lower differentiation and high metastasis, it usually has metastasis in cervical lymph node and/or distant sites. Recurrence and metastasis lead to the failure of treatment. Multi-genes and multi-factors are involved in the processes of NPC recurrence and metastasis. Elucidating molecular mechanisms of NPC recurrence/metastasis and searching for effective target therapies are the promising pathway to improve the survival.Recently, epithelial-mesenchymal transition (EMT), the fundamental processes regulating morphogenesis in multicellular organisms, is thought to play a key role during early steps of invasion and metastasis of epithelial malignancies. Tumor metastasis involves a sequential series of processes which promote and regulate the escape of migratory cancer cells to generate metastatic lesions at distant sites. The process begins in the primary tumor, where tumor cells dysregulate homotypic cell adhesion, downregulate cell adhesion proteins such as E-cadherin, and upregulate proteins characteristic of a more motile, mesenchymal-like phenotype such as vimentin. This process requires transcriptional reprogramming to suppress E-cadherin expression via transcription factors associated with EMT.Epidermal growth factor receptor (EGFR), a member of the ErbB receptor tyrosine kinase family, is a single pass transmembrane tyrosine kinase. Signaling through EGFR induces proliferation, invasion, and angiogenesis of tumor cells. Three major signaling pathways mediate the downstream effects of EGFR activation at the cellular level:(1) the Ras-Raf-MAPK kinase pathway, (2) PI3K-Akt pathway, and (3) JAK/STAT pathway. And PI3K/Akt pathway is core features of EMT.E-cadherin is a cell-cell adhesion molecule and calcium-dependent transmembrane glycoprotein present in most epithelial cells in embryonic and adult tissues, which has previously been found to be expressed in well-differentiated non-invasive carcinoma cell lines but is lost in many poorly differentiated invasive cell lines. Loss of E-cadherin is a hallmark ofEMT.Study about the EGFR signaling way, E-cadherin and EMT in NPC was not reported simultaneously before. In this research, we studied on weather EGFR signaling way can induce EMT by downregulating the expression of E-cadherin in NPC.Objective1. To investigate the expression of EGFR, p-EGFR and E-cadherin in NPC tissues, and research on the relationship between them and clinicopathology parameters in NPC respectively;2. To understand the expression of EGFR, p-EGFR and E-cadherin in NPC cell lines, study on the effect and research on the relationship between them and cell motility, invasion and metastases respectively;3. To investigate weather EGFR signaling way can induce EMT and enhance cell motility, invasion and metastases by regulating the expression of E-cadherin in NPC.Methods1. Studied on the expression of EGFR, p-EGFR and E-cadherin in NPC tissues.54 cases of NPC tissues were collected to be studied and 23 cases of nasopharyngeal chronic inflammation tissues were used as control.(1) The expression of EGFR, p-EGFR and E-cadherin were determined by immunohistochemistry;(2) The relationship between them and clinicopathology parameters were studied.2. Detected the expression of EGFR, p-EGFR and E-cadherin protein, as well as cell motility, invasion and metastases in NPC cell lines.NPC cell lines 5-8F, CNE-1, CNE-2 and 6-1 OB were treated respectively.(1) Wound healing assay:detected cell motility and compare the difference between them;(2) Transwell invasion assay:detected cell invasion and metastases;(3) Western blot:examined the expression of EGFR, p-EGFR and E-cadherin protein; (4) Investigated the relationship between the expression of EGFR, p-EGFR, E-cadherin and cell motility, invasion and metastases.3. Investigated the impact of epidermal growth factor receptor (EGFR) ligand EGF and small molecule tyrosine kinase inhibitor Erotinib on the phenotype, cell motility, invasion and metastases in NPC cell lines.The highest and lowest expression of EGFR cells were selected as the objects.(1) MTT assay:measured cell proliferation in different groups;(2) Inverted microscope:observed the cell phenotype changes;(3) Wound healing assay:detected the changes of cell motility;(4) Transwell invasion assay:detected the changes of cell invasion and metastases;(5) Western blot:examined the changes of the expression of EGFR, p-EGFR, Vimentin and E-cadherin protein;(6) RT-PCR:examined the changes of the expression of EGFR mRNA, and E-cadherin mRNA.Results1. The active expression rate of EGFR in NPC tissues was 68.5%(37/54), and p-EGFR was 61.1%(33/54), and E-cadherin was 35.2%(19/54); The active expression rate of EGFR in nasopharyngeal chronic inflammation tissues was 34.8%(8/23), and p-EGFR was 21.7%(5/23), and E-cadherin was 91.3%(21/23); the active expression rate of EGFR and p-EGFR in NPC were significantly higher than the nasopharyngeal chronic inflammation (p<0.05), the active expression rate of E-cadherin in NPC were significantly lower than the nasopharyngeal chronic inflammation(p<0.05);2. The expression of EGFR were significantly correlated with lymph node metastases(P<0.05), and the differences of EGFR expression between the different age, gender, T stage, clinic stage and distant metastases were not statistically significant(p>0.05); the differences of p-EGFR expression between the different age, gender, T stage, clinic stage, lymph node metastases and distant metastases were not statistically significant(p>0.05); the expression of E-cadherin were significantly correlated with clinic stage, lymph node and distant metastases (P<0.05), the differences of E-cadherin expression between the different age, gender, and T stage were not statistically significant (p>0.05);3. The expression of EGFR was positive correlated with the expression of p-EGFR (r=0.532, p<0.05); the expression of EGFR was negative correlated with the expression of E-cadherin (r=-0.345, p<0.05); the expression of p-EGFR was no correlated with the expression of E-cadherin (r=-0.122, p=0.380);4. In NPC cell lines,5-8F had the strongest ability of motility, invasion and metastases than others(p<0.05); 6-1OB had the weakest ability of motility, invasion and metastases than others(p<0.05); the expression of EGFR and p-EGFR was the highest in 5-8F(p<0.05), the lowest in 6-10B(p<0.05); the expression of E-cadherin was the lowest in 5-8F(p<0.05), the highest in 6-10B (p<0.05);5. The expression of EGFR and p-EGFR were positive correlated with cell motility, invasion and metastases; the expression of E-cadherin was negative correlated with cell motility, invasion and metastases;6. In the presence of EGF treatment,5-8F cells changed its shape to fusiform fibroblastoid phenotype and its colony formation from compact to sparse; EGF promoted the wound healing(p<0.05), resulted in the increased cell motility and movement(p<0.05), induced up-regulation of vimentin and p-EGFR protein and down-regulation of E-cadherin protein and mRNA expression in 5-8F cells(p<0.05);7. Erlotinib induced the cell morphological changes to round or square, cell grew into cluster, delayed the wound healing, and partly inhibited cell motility and movement(p<0.05), down-regulate of vimentin and p-EGFR protein, up-regulate the E-cadherin protein and mRNA expression in 5-8F cells(p<0.05);8. In the presence of EGF or Erlotinib treatment, there is no significant changes in phenotype, motility, movement and expression of protein as well as mRNA in 6-10B cells(p>0.05).Conclusion1. EGFR signaling pathway and E-cadherin played different roles in the tumor occurrence and development; 2. The abnormal expressions of EGFR and E-cadherin may play a roles in N stages and M stages of NPC;3. EGFR signaling way may induce EMT, and enhance invasion and metastases of NPC cells by down-regulating of E-cadherin;4. The sensitivity of NPC cells to EGFR inhibitor is mainly dependent upon the expression of EGFR;5. EGFR inhibitor may influence the course of EMT in NPC by up-graduation of E-cadherin, and then decrease the abilities of metastasis of NPC. |
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