CD166 Mediated EGFR Phosphorylation Promote The Growth Of Oral Squamous Cell Carcinoma

Background & Purpose: In a previous study,we compared the membrane proteomics between cancer stem like cells and non stem like cells derived from HNSCC,and identified Activated Leukocyte Cell Adhesion Molecule(CD166 or ALCAM)as a relative specific cell surface marker for cancer stem like cells in HNSCC,for it not only increasingly expressed in cancer stem like cells,its expression level was significantly related to malignant behavior of HNSCC cells both in vivo and in vitro?Our previous studies showed that compared with well recognized stem cell related cell surface marker CD44,membrane expression of CD166 is a more effectively stem like cell enrichment marker in head and neck cancer.However,till now,very limited data related to the function of CD166 in malignant cancer and how it involves the stemness trait has been reported.In this study,we tried to identify EGFR as one interaction protein of CD166 in head and neck cancer,and more importantly,we found that CD166 influence EGFR phosphorylation and its downstream signaling molecule FAK to coordinately regulate EGF-induced cell migration by inhibiting cbl-1 mediated ubiquitination and degradation of phosphorylated EGFR.Methods: expression of CD166 mRNA was examined in 40 fresh tumor samples and paired negative resection margins by real-time PCR.expression of CD166 protein levels was examined in 20 fresh tumor samples and paired negative resection margins by Western blot?Through cultivation of microspheres,we detected the expression of CD166 in RNA and protein levels.To detect the function of cd166 in HNSCC: the growth curve,invasion assay,clony formation,sphere formation assay and vivo tumorigenicity.By Confocal Microscope Analysis,The double immunofluorescent staining revealed if there is co-localization of cd166 and EGFR in HN13 cells..EGFR was identified as one of the CD166 interaction proteins identified by immunoprecipitation(IP)and MS.Then to further test the interaction between EGFR and CD166 influence the activation of EGF/EGFR signaling.To investigate the clinical value of membrane co-localization and interaction between EGFR and CD166,we used tissue arrays to examine the expression of CD166 and EGFR phosphorylation(Tyr 1068)in HNSCC clinical samples and analyzed the relationship.To test function of CD166 on the HNSCC was mediated by activation of EGF/EGFR signaling,its down stream phosphorylation of FAK and re-organization of cytoskeletal by Confocal Microscope Analysis.Results: The mRNA level in tumors was significantly higher than in negative margins(P=0.0011).Then,expression of CD166 protein was examined in 20 fresh tumor samples and paired negative resection margins by Western Blot.CD166 protein levels in tumor tissues were generally much higher than in negative margins.The expression level of CD166 in sphere culture and adherent culture was also compared,both CD166 m RNA and protein levels were remarkably increased in sphere culture spheres than their traditional adherent culture counterparts.CD166 played important functions in sphere formation ability,colony formation ability and invasion ability of HNSCCs?The endogenous interaction between EGFR and CD166 was further confirmed by co-immunoprecipitation.Co-localization of CD166 and EGFR on cell membrane was also detected in both HN13 and HN30 cells.In both HN13 and HN30 cells,decreasing CD166 expression resulted in inhibiting the activation of EGF/EGFR signaling after EGF treatment;while increasing CD166 expression led to enhancing activation of EGF/EGFR signaling,In transient CD166 overexpression HN13 and HN30 cells,significantly enhanced activation of EGF/EGFR signaling was observed after EGF treatment,as indicated by strongly increasing expression of p EGFR(Tyr1068?Tyr1045),p AKT,p FAK and p ERK.suggesting that signaling downstream of EGF/EGFR was promoted by CD166.The staining percentage value of CD166 and p EGFR was positively associated with each other in these 87 cases,with R=0.373 and p<0.001 with spearman correlation analysis.CD166 stabilizes p EGFR and decreases ubiquitinated forms of p EGFR after EGF stimulation.CD166 enhanced colony formation ability and invasion ability of HNSCC cells was mediated by activation of EGF/EGFR signaling,its down stream phosphorylation of FAK and re-organization of cytoskeletal.Down-regulation of CD166 inhibited tumor growth in vivo.Conclusion: In this study,we have identified a novel function of CD166 in stabilization of active form of EGFR and enhanced the activation of EGF/EGFR signaling by interaction with EGFR.Our data suggested that CD166 enhanced EGF/EGFR signaling activation and promoted the phosphorylation of FAK,leading to active re-organization of cell skeletal after EGF stimulation and increasing colony formation ability and invasion ability of HNSCC cells,while knockdown of CD166 can promote EGFR ubiquitination and subsequently degradation of active form of EGFR,following with rapidly inactivation of EGF/EGFR signaling.CD166 might be an attractive therapeutic target in HNSCC treatment.