Signal Pathway And Function Of EGFR-regulated Proteins-APP And Cystatin C In Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma(NPC) is a rare malignancy in most part of the world and it is one of the most confusing, commonly misdiagnosed and poorly understood diseases. The cancer is an Epstein-Barr virus-associated malignancy with a remarkable racial and geographical distribution. It is highly prevalent in east-southem Asia where the disease occurs at a prevalence about a 100-fold higher compared with other populations not at risk. epidermal growth factor receptor (EGFR, HER1) is overexpressed in a majority of nasopharyngeal carcinoma(NPC). Their abnormally high expression signifies poor prognosis inNPC patients. EGFR silencing by RNAi can reduce the proliferation of nasopharyngeal carcinoma cells and induce cell cycle arrest at G1 phase.In order to reveal the mechanism of NPC carcinogenesis regulated by EGFR. In our laboratory we cultured NPC cell line-CNE2 in serum-free medium to deplete contaminate from bovin serum, and stimulated cell in experimental group with TGF-αfor 24 hours, proteins secreted from NPC cell lines were seperated by two-dimensional electrophoresis(2-DE), analyzed by PDquest and MALDI-TOF MS, We found 8 differential expression proteins between the control group and experimental group. These proteins present EGFR-regulated secreted proteins. Among them Amyloid beta A4 protein and Heterogeneous nuclear ribonucleoprotein A1 was up-regulated, while TPM4-ALK fusion oncoprotein type 2, 14-3-3 protein epsilon isoform transcript variant 1, Annexin AS, Metalloproteinase inhibitor 2, Cystatin C and 14-3-3 protein zeta/delta were down-regulated after TGF-αtreatment.Abnormal cleavage of amyloid precursor protein(APP) in the central nervous system has been linked to the development of Alzheimer's disease. Some work has identified additional roles for APP in peripheral tissue, such as cellular proliferation and motility. Some investigations have identified the up-regulation of APP expression in pancreatic cancer cells both in vitro and in vivo. And secretory APP(sAPP)can be detected in collected medium in vitro. Inhibition of sAPP signaling reduces pancreatic cancer cell number via a reduction in cellular proliferation. Cystatin C(Cyst C) is a secreted cysteine protease inhibitor. Cyst C is ubiquitously expressed and present in most bodily fluids where it inhibits the activities of cathepsins, a family of cysteine proteases that can promote cancer cell invasion and metastasis.Independent of its effects on cathepsin activity, Cyst C also regulates cell proliferation, raising the possibility that CystC targets protease dependent and-independent pathways. Cyst C complex concentrations were suggested as diagnostic and prognostic indicators for cancers of the skin, colon, and lung. In this study, APP and cystatin C were selected for further analysis, At first, we detected the expression of EGFR,APP and Cys C by Western blotting and Immunohistochemistry. To investigate the correlation between EGFR and the other two proteins and the potential significance of EGFR, APP and cys C in the development of nasopharyngeal cancer. the results showed that EGFR was overexpressed in the tumor tissues including the primary nasopharyngeal cancer tissues and metastasis in the neck lymph node. In a fact, we also found that EGFR expression was higher in the neck metastasis lymph node than that in the nasopharyngeal cancer tissues (P＜0.05). At the same time we found APP expression is higher in the tumor tissues than Nasopharyngeal inflammation mucous tissue, but no difference between the primary nasopharyngeal cancer tissues and metastasis in the neck lymph node. Cystatin C expression is lower in the tumor tissues than Nasopharyngeal inflammation mucous tissues, but no difference between the primary nasopharyngeal cancer tissues and metastasis in the neck lymph node. Analyse the correlation between EGFR and the other two proteins, the results showed that EGFR expression was positively related to APP expression (r=0.586, P＜0.01); EGFR expression was negatively related to Cys C expression (r=-0.396, P＜0.01)To determine the signaling pathway involved in activation of amyloid precursor protein and Cystatin C expression regulated by EGFR in CNE2, we treated the cells with special inhibition of EGFR(PD153035),the PI3 kinase pathway(wortmannin) and the MAP kinase pathway(PD98059), respectively. As a result, abrogation of the PI-3 kinase drastically altered the expression of APP and Cystatin C in CNE2 cell. In contrast, inhibition of the MAP kinase pathway didn't affect their levels obviously. It was indicated that APP and Cystatin C might be mainly regulated through the PI3K signal pathway, but the MAPK signal pathway also participatied in the adjustment. For the function of sAPP in CNE2 cell, we did MTT and the Wounding Assay experiment and found the cellular proliferation is weaken, the movement is also weaken after neutralization of sAPP.In summary, This research confirms that in nasopharyngeal carcinoma APP and cys C protein are truly EGFR-regulated proteins through the western blot and the immunohistochemistry methods. And discovered APP may promote the cell proliferation and movement. In the NPC occurrence and development EGFR regulated APP and cys C expression possibly mainly through the PI3 kinase pathway to display their functions.