The Role Of TrkB Expression In EMT Induction In Nasopharyngeal Carcinoma And Its Relationship With Tumor Invasion And Metastasis

Nasopharyngeal carcinoma (NPC) is one of the most common cancers among people of Chinese, especially in southern China. The most common pathological type of NPC is low differential squamous cell carcinoma, which is almost invariably accompanied by metastasis, accounting for the most common cause of death of cancer patients. Besides, the5-years survival rate is between41-75%.It is well known that the epithelial-mesenchymal transition (EMT) play an important role in the first step of metastasis. Epithelial cells acquire properties reminiscent of those of mesenchymal cells, which actively downregulate cell-cell adhesion systems, lose polarity, and finally reduced intercellular interactions and increased migratory during EMT. Several epithelial proteins are downregulated, such as E-cadherin, catenin. But mesenchymal proteins, including vimentin, fibronectin and smooth muscle actin, can be upregulated. Furthermore, E-cadherin is the best-characterized molecular marker of EMT and loss of E-cadherin expression is an EMT hallmark.Tropomysin-related kinase B (TrkB) is a member of Trk family, functions as a receptor tyrosine kinase. Signaling through TrkB induces hypoxic response, cellular motility, and anoikis detachment-induced apoptosis inhibition. When activated by BDNF, TrkB leads to the activation of downstream signaling molecules, such as Ras/MAPK pathway and phosphoinositide-3kinase/protein kinase B (PI3K/Akt) pathway which differential induce the regulation of apoptosis and metastasis. Recent reports indicated that TrkB could participate in the process of EMT.To our knowledge, the relationship between TrkB and EMT in NPC has not been sufficiently evaluated. In this study, we investigated the association between TrkB expression and NPC clinical outcome and whether TrkB signaling way can regulates EMT by downregulating the expression of E-cadherin in CRC cell.Objective1. To investigate the expression of TrkB and E-cadherin in NPC tissues, analyze the association between them and the links between TrkB expression and NPC clinical outcomes.2. To investigate the expression of TrkB and E-cadherin in human NPC cell lines, analyze the association between them, and study on the effect of TrkB on invasion and metastasis.3. To investigate whether TrkB signaling way could induce EMT by regulating the expression of E-cadherin in NPC cells in vitro.4. To investigate weather K252could inhibit TrkB signaling way and the growth of implanted tumor in nude mice.Methods1. Expression and clinical significance of TrkB and E-cadherin in NPC tissue. In this part, we adopted immunohistochemistry to investigate the expression of TrkB and E-cadherin in NPC tissue and analyzed the association between them. Pertinent clinical features and specimens of tumor were obtained from54patients who were diagnosed as NPC. Other25normal patients were used as control.2. Expression of TrkB and E-cadherin in NPC cell lines and the association between their expression and the biological behaviors. Nasophaiyngeal epithelial cell line NP69, nasopharyngeal carcinoma cell lines5-8F,6-10B and laryngeal cancer cell line Hep-2were treated respectively. We adopted Western blot assay to investigate the expression of TrkB and E-cadherin in different cell lines. Transwell invasion assay and Wound healing assay were used to detect cell motility, invasion and metastases, respectively.3. Impact of BDNF,K252a on the invasion and metastases in human nasopharyngeal carcinoma cell lineHuman nasopharyngeal carcinoma cell lines cell5-8F was treated with200ng/ml BDNF and400nM K252a respectively. Investigate cell proliferation in different groups by MTT assay. Expression of TrkB and E-cadherin were examined by Western blot and RT-PCR. The invasion and metastases were analyzed by Wound healing assay and Transwell invasion assay. 4. Impact of K252a on the growth inhibition, cell motility in node mice. We constructed nude mice sub-skin model transplanted with nasopharyngeal carcinoma cell, investigated whether K252could inhibit and the growth of implanted tumor. The expression of TrkB and E-cadherin in implanted tumor was examined by Western blot assay.Results1. In NPC tissue, the mean staining score of TrkB was137±39and that of E-cadherin was156±50.The difference of mean staining score between NPC tissue and normal nasopharyngeal tissue were statistically significant(p<0.001)2. The expression of TrkB were significantly correlated with lymph node metastases (p<0.001) and clinic stage (p=0.007). The differences of TrkB expression between the different age (p=0.66), gender (p=0.209) and T stage (p=0.622) were not statistically significant;3. The expression of E-cadherin were significantly correlated with lymph node metastases (p<0.001) and clinic stage (p=0.001). The differences of TrkB expression between the different age (p=0.328), gender (p=0.149) and T stage (p=0.298) were not statistically significant;4. The expression of E-cadherin was positive correlated with the expression of TrkB(p<0.001)5. TrkB staining score could also be the statistically significant predictors of lymph node metastases;6. We classified patients as TrkB low-expression group and TrkB high-expression group according to the mean staining score. The5-year survival rate in TrkB high-expression group was significantly reduced than that of TrkB low-expression group (Log-rank,p=0.042);7. We classified patients as E-cadherin low-expression group and E-cadherin high-expression group according to the mean staining score. The5-year survival rate in E-cadherin high-expression group was significantly increased than that of E-cadherin low-expression group (Log-rank,p=0.047)8. Normal nasopharyngeal cell did not express TrkB, but expressed E-cadherin. The difference between the expression of TrkB or E-cadherin in nasopharyngeal cell line5-8F and6-10B was statistically significant (p<0.001). Laryngeal cancer cell line Hep-2express TrkB, too. TrkB was not the specific indicator of NPC cell.9. The expression of TrkB was positive correlated with the expression of E-cadherin in NPC cell lines(r=0.987,p<0.001).10.The expression of TrkB was positive correlated with cell motility, invasion and mtastasis; The expression of E-cadherin was positive correlated with cell motility, invasion and mtastasis; 11.In the presence of BDNF treatment,5-8F cells changed its shape to fusiform fibroblastoid phenotype. BDNF promoted the wound healing (p<0.05) and increased cell motility, induced up-regulation of TrkB and vimentin and down-regulation of E-cadherin protein and mRNA expression.(p<0.05)12.K252a induced the cell morphological changes to round or square, cell grew into cluster, delayed the wound healing, and partly inhibited cell motility(p<0.05), down-regulate of vimentin and TrkB protein, up-regulate E-cadherin protein and mRNA expression.(p<0.05);13.K252a inhibited the growth of implanted tumor. Western blot assay demonstrated that K252a down-regulate the expression of TrkB and dowen-regulate the expression of E-cadherin in implanted tumor of nude mice.Conclusion1. TrkB overexpression was observed in NPC tissue and it was collected with lymph node metastases,clinic stage and poor outcomes.2. TrkB signaling way could induce EMT and enhance invasion and metastasis of NPC cells by down-regulating of E-cadherin.3. K252a may influence the course of EMT and decrease the abilities of metastasis in NPC.