The Functions Of Tyrosine Kinase Receptor EphB4 And Its Interacting Partner RACK1 In The Tumorigenesis Of Esophageal Squamous Cell Carcinoma

Part ?Down-regulation of EphB4 phosphorylation is necessary for the tumorigenesis of esophageal squamous cell carcinomaObjective: Eph receptors,the largest subfamily of transmembrane tyrosine kinase receptors,have been increasingly implicated in various physiological and pathological processes,and the roles of the Eph family members during tumorigenesis have recently attracted growing attentions.Until now,EphB4 has been reported to play an important role in the progression of various cancers,including breast cancer,bladder cancer.However,the function of EphB4 and its ligand ephrin B2 in the carcinogenesis of esophageal squamous cell carcinoma is not fully understood.Method: The expression of EphB4 and ephrin B2 in ESCC tissues,paired normal tissues and cell lines were examined using Realtime PCR and western blot;ephrin B2-Fc was purified and its effects on cell growth and migration were examined using MTT assay,soft agar assay and Boyden Chamber assay;the effects of EphB4 and EphB4 KD on the growth and migration of were investigated by forcing the expression of EphB4 and EphB4 KD in ESCC cell lines;the interaction between RACK1 and EphB4 was studied using immunostaing,immunoprecipitation and GST-pull down assay.Result: Here,it was found that the expression of EphB4 was up-regulated in ESCC(esophageal squamous cell carcinoma)cell lines and cancer tissues compared with the paired normal tissues,while ephrin B2 was down-regulated in ESCC samples.Phosphorylation of EphB4 induced by its ligand ephrin B2-Fc inhibited the growth,migration and colony formation of ESCC cells.Moreover,over-expression of EphB4 or EphB4 kinase dead mutant(EphB4 KD)in ESCC cells promoted cell growth and migration,suggesting EphB4 promoted cell growth and migration independent of its kinase activity.Furthermore,in the yeast two-hybrid screening for EphB4 interacting protein,we found that EphB4 interacted with the adaptor protein RACK1 and RACK1 decreased the phosphorylation level of EphB4.Conclusion: Taken together,our study revealed the important function and regulation of EphB4 phosphorylation in the progression of ESCC and suggested EphB4 as a novel target for the treatment of ESCC.Part? RACK1 promoted the growth and migration of the cancer cells in the progression of esophageal squamous cell carcinomaObjective: Receptor for activated protein kinase C(RACK1)is an adaptor protein with seven WD40 repeats.Through binding a large number of signaling proteins,RACK1 has been involved in multiple intracellular signal pathways.With regard to its roles,RACK1 coordinated cell growth,movement,metabolism and multiple biological processes.However,the function of RACK1 in ESCC remains largely unknown.Method: The expression of RACK1 in ESCC tissues and paired normal tissues was examined using Realtime PCR and western blot;RACK1 was overexpressed or knocked down in ESCC cell lines and the effects of RACK1 on cell growth and migration were examined using MTT assay,soft agar assay and Boyden Chamber assay;the effects of RACK1 on the metastasis of ESCC cells were investigated using an in vivo system;the effects of RACK1 on the activity of Hedgehog signaling and the expression of Hedgehog signaling target genes were studied using reporter assay,RT-PCR,and Boyden Chamber assay.Result: We have found the novel function of RACK1 independent of EphB4 in the progression of ESCC.Here,we found that the expression of RACK1 was up-regulated in ESCC clinical samples with the help of Realtime PCR and Western Blot.Moreover,over-expression of RACK1 in ESCC cells promoted cell proliferation and migration,while down-regulation of RACK1 impaired the proliferation and migration of ESCC cells in vitro and in vivo.Mechanistically,RACK1 was a positive regulator of Hedgehog signaling and promoted the proliferation and migration of ESCC cells by activating hedgehog signaling.RACK1 increased the expression of Gli1 and Patch,two target genes of Hedgehog signaling,while down-regulation of RACK1 decreased the expression of Gli1 and Patch.Moreover,knockdown the expression of Gli1 rescued the function of RACK1 in ESCC cells.Conclusion: Taken together,our study revealed that RACK1 promoted the growth and migration of ESCC cells by activating Hedgehog signaling.