Targeting Insulin-like Growth Factor-1Receptor In Nasopharyngeal Carcinoma

Experiment part one:Expression and Clinical Significance of Insuline like Growth factor-1Protein in Nasopharyngeal carcinomaObjective To evaluate the expression of IGF-1R protein and its clinical signification in nasopharyngeal carcinoma. Methods S-P immunohistochemical staning was performed to detect the expression of IGF-1R in63cases of NPC and21inflamatory nasopharyngeal mucosa; and the expression of IGF-1R in three NPC cell lines and chronic nasopharngotis tissues was examined by western blots. Results The expression level of mRNA and protein of IGF·1R in tumor samples were significantly higher than in inflamatory nasopharyngeal mucosa tissue.(1)The positive rates of IGF-1R were82.5%and14.3%respectively in63cases of NPC and21inflamatory nasopharyngeal mucosa;(2)It also showed stronger positive expression in Stage Ⅲ-Ⅳ of NPC than that in stage Ⅰ-Ⅱ (P<0.01);(3)IGF-1R positive expression in cases of NPC with cervical lymph node metastasis was significantly higher than that of NPC without lymph node metastasis(P<0.05);(4) it more often appears in recurrent cases than in primary cases(P<0.05);(5) IGF-1R positive expression had a significantly stronger in patients whose survival time less than5years than that in patients whose survival time over5years (P<0.01). Conclusion Over-expression of IGF-1R is related to poor differentiation of NPC, also may be linked with oncogenesis and development of NPC. The over-expression of IGF-1R may be one of a prognostic factors in NPC. The IGF-1R might be a potential therapeutic target in nasopharyngeal cancer.Experiment part two:The Expression of insulin-like growth factor-1receptor,the Activation of PI3K/AKT and ERK/MAPK signal transduction pathway in Nasopharyngeal Cancer cellsObjective:The Insulin-like growth factor I (IGF-I) receptor can induced activation of downstream effector molecules,such as phosphatidylinositol3-kinase(PI3K)/AKT and mitogen-activated protein kinase(MAPK),which plays an important role in cell survival,prevention of apoptosis, proliferation and differentiation of some human malignancies. Objective To study the expression of IGF-1R and its downstream pAKT/AKT, p-ERK/ERK1/2and the effect of treatment with PI3K/AKT and ERK/MAPK specific inhibitors in the human nasopharyngeal cancer cells and to evaluate the relationships among them. Methods Protein expressions of IGF-1R and its downstream p-Akt/Akt,p-ERK/Erk in human nasopharyngeal cancer cell line CNE2were examined by western blots and the regulations of IGF-1stimulation, PI3K/AKT and ERK/MAPK specific inhibitors, Wortmannin and PD98059on this cell. Results:Protein levels for IGF-1R, Akt和Erkl/2were significantly higher in nasopharyngeal cancer cell line than in control nasopharngotis tissues. The expression of pAKT/AKT and p-ERK/ERKl/2were strongly associated with overexpression of IGF-1R. IGF-1stimulation induced more p-ERK1/2and p-Akt activation in nasopharyngeal cancer cell line (CNE2) than that in control(without IGF-1). Treatment with PD98059or Wortmannin could block activation of p-ERK1/2or p-Akt in nasopharyngeal cancer cell line (CNE2). Conclusion:Our study shows that the expression of IGF-1R and the activation of pAKT and pErkl/2pathway may play an important role in the pathogenesis of nasopharyngeal cancer. IGF-1was one of the important factors to activate PKB/AKT and ERK/MAPK signal transduction pathway.The IGF-1R system might be a potential therapeutic target in nasopharyngeal cancer.Experiment part three:A novel treatment approach in Human Nasopharyngeal Carcinoma cells:Picropodophyllin, a specific inhibitor of the Insulin-like Growth Factor-1Receptor Objective:Nasopharyngeal carcinoma (NPC) is the most common head and neck cancer in southern China and South East Asia. IGF-1R has represented a promising new therapeutic target for anticancer therapy. IGF-1R was over-expressed in most of carcinoma tissues and was associated with poor prognosis, but their expression in NPC remains unclear. Methods:Four NPC-derived cell lines,CNEl,CNE2,TW03and TW03Epstein-Barr virus (EBV)(+) were examined and the effect of IGF-1R specific tyrosine kinase inhibitors-PPP were studied in these cell lines.Results:IGF-1R protein expression was highest in the TW03cell line, moderate in CNE2and CNE1, and lowest in TW03EBV(+). On two main downstream signaling pathways(PI3K and MAPK) of IGF-1R activation,PPP can distinctly reduced protein levels of phosphorylated Akt and induced apoptosis cells of CNE1,CNE2, TW03and TW03EBV(+) cell lines, it just slightly decreased levels of phosphorylated ERK1/2;efficiently blocked phosphorylated IGF-1R activity in CNE1,CNE2and TW03three cultured tumor cell lines,and cell lines with high IGF-1R expression responded promptly;In addition, obviously displayed growth inhibitory activity against NPC cells in these cultured cell lines; but these responses were very weak in TW03EBV(+).These data implied that infection of EBV promotes the tumorigenicity of NPC cells and reduces effect of anti-tumor therapy,meanwhile, it may be involved in degradation of IGF-1R protein. Conclusions:Our data provide evidence that IGF-1R is a promising target for treatment of NPC.Experiment part four:Experiment part four:Effect of β-arrestinl in Internalisation and Ubiquitination of Insulin-like growth factor-1receptor(IGF-1R)Insuline like growth factor-1receptor(IGF-1R) has been shown to be crucial for tumor growth,transformation,development and prevention of apoptosis,and has emerged as a general and promising target for cancer therapy. Internalization and ubiquitination of the IGF-1R has been pay attention to.Objective To investigate if internalization of the IGF-1R is required for other functions apart from down-regulation of the receptor and to examine the role of β-arrestin in internalization of the IGF-1R. Methods:P6, β-arrestinl positive(WTl) and β-arrestinl negative, IGF-1R internalization of three cell lines were examined by western blots,immunoprecipitation. Results In P6cells an obvious decrease in the level of un-internalised IGF-1R(surface-bound IGF-1R) could be seen as ligand stimulation time increases;but a clear and distinct increase in internalization of the IGF-1R could be seen. In β-arrestinl negative cells the IGF-1R bands could not be seen,indicating the absence of internalized IGF-1R,the positve control band could however be seen;Un-internalised IGF-1R bands could be seen,its level remain constant. Examine activation of PI3K and MAPK pathways,in the absence of β-arrestinl the level of phosphorylation of pERK was not detected when the ligand stimulated the IGF-1R. Conclusion Our results clearly demonstrated that IGF-1R internalization could not occur in the absence of β-arrestinl.Using β-arrestinl as a tool to modulate IGF-1R internalization,and IGF-1R internalization is required for activation of one of the major intracellular signaling pathways,the MAPK pathway.