The Function And Mechanism Of AURKA-SDCBP-EGFR Axis In Esophageal Squamous Cell Carcinoma

Background and research purposeEsophageal squamous cell carcinoma(ESCC)is the most important pathological type of esophageal cancer,accounting for ninty percent of esophageal cancer.It is a complex disease with multiple etiologies.ESCC is often diagnosed at an advanced stage and is prone to drug resistance,metastasis and relapse.Although multidisciplinary treatment methods including surgery,chemotherapy,and radiotherapy have been widely used in the treatment of ESCC,the five-year survival rate of ESCC patients is still less than 20%.The limited effect of traditional therapies on ESCC promotes us to discover a new mechanism during the development of ESCC,and apply it as a target for the diagnosis and treatment of ESCC.Aurora kinase A(AURKA)belongs to the serine/threonine protein kinase family and plays an important role in the regulation of cell mitosis.As a protein kinase,AURKA modulates the function of its substrates by phosphorylating the substrates.More importantly,AURKA plays an important role in tumor formation and promotes tumorigenesis in many types of cancer.At present,many AURKA-specific inhibitors or inhibitors for the Aurora kinase family have been developed,and some have been used in clinical trials,especially the AURKA-specific inhibitor Alisertib has been applied in the phase Ⅲ clinical trial,indicating that AURKA is an important and meaningful target for cancer.In ESCC,there are several reports that the expression of AURKA is elevated in cancer tissues of ESCC patients,and the expression of AURKA is related to the poor prognosis of patients.Functionally,AURKA can promote the proliferation,invasion and migration of ESCC cells.Although AURKA is highly expressed in ESCC and has a cancer-promoting effect,the mechanism of AURKA,especially the substrate protein of AURKA in ESCC,is rarely studied.The deficiencies of these studies hinder the further application of AURKA inhibitors in ESCC.In this project,we screened and verified syndecan binding protein(SDCBP)as a substrate protein of AURKA in ESCC through His-pull down and liquid-phase mass spectrometry techniques.We further studied the mechanism how AURKA regulate SDCBP.SDCBP is a scaffold protein containing PDZ domain,which regulates downstream pathways and cell functions by mediating the interaction between proteins.Although SDCBP can promote cell proliferation,invasion and metastasis,regulate stemness of cancer cells,and promote angiogenesis in a variety of cancers,the role and mechanism of SDCBP in ESCC have not yet been reported.This study screened and verified that SDCBP is the substrate protein of AURKA in ESCC through His-pull down and liquid-phase mass spectrometry.This study further studied the expression and function of SDCBP in ESCC,as well as the downstream signaling pathway of SDCBP,and proved that SDCBP is functional downstream of AURKA.Thus,this project proved that targeting the AURKA-SDCBP axis in ESCC is of great significance.Based on the above research background and research ideas,this project was performed from the following four parts:1)Screen SDCBP as the downstream substrate protein of AURKA in ESCC;2)Explore whether AURKA phosphorylates SDCBP and how AURKA regulates SDCBP after phosphorylation of SDCBP;3)Study the expression and function of SDCBP in ESCC;4)Study the downstream signaling pathway of SDCBP in ESCC.Methods1.Screen downstream substrate of AURKA in ESCCAURKA protein was purified in prokaryotic expression system with NI-NTA beads.The His-pull down experiment and liquid phase mass spectrometry technology were used to analyze and identify proteins that can interact with AURKA in ESCC cells.Functional enrichment analysis of these proteins was performed with David database.Endogenous co-immunoprecipitation experiments was performed to confirm the potential AURKA-interacting proteins from mass spectrometry data.Endogenous and exogenous co-immunoprecipitation experiments were performed to verify the binding ability between AURKA and SDCBP.Western blot and immunohistochemistry were used to detect the correlation between the expression of AURKA and SDCBP in ESCC cell lines and patient tissues.2.Explore the phosphorylation modification of SDCBP by AURKAPurify SDCBP protein in the prokaryotic expression system using NI-NTA beads,and test whether AURKA can phosphorylate SDCBP by in vitro protein kinase assay.Predict the possible phosphorylation sites of SDCBP based on the the Netphos2.0 website and AURKA phosphorylation consensus motif.Construct these SDCBP phosphorylation site inactivation mutation plasmids and clone them into pet28a vector.Using NI-NTA beads to purify the SDCBP phosphorylation site inactivation mutation proteins in the prokaryotic expression system.The in vitro protein kinase experiment was used to detect the phosphorylation of AURKA on the phosphorylation site inactivation SDCBP protein compared with the wild-type SDCBP protein.3.Study the regulation mechanism of SDCBP by AURKAThe expression of mRNA and protein levels of SDCBP after AURKA knockdown or overexpression was detected by quantitative PCR and Western blot.Western blot was used to detect the effect of AURKA on the stability of SDCBP protein after cycloheximide(CHX)treatment,and the effect of phosphorylation at Ser131 and Thr200 sites of SDCBP on the stability of SDCBP protein.Coimmunoprecipitation assay was performed to detect the effect of AURKA on the ubiquitination of SDCBP protein,and the effect of SDCBP phosphorylation on the ubiquitination of SDCBP protein.4.Investigate the expression and function of SDCBP in ESCCThe expression of SDCBP in ESCC patient cancer adjacent tissues and cancer tissues was detected by immunohistochemistry.The correlation between SDCBP expression in cancer tissues and clinicopathological data was analyzed.The proliferation and colony formation ability of ESCC cells was detected after knockdown or overexpression of SDCBP,or after inactivation of SDCBP phosphorylation,by MTT and soft agar colony formation assays.The cell derived xenograft(CDX)model was used to study the tumor growth after knocking down SDCBP.Rescue assay was performed to detect whether SDCBP is a functional downstream molecule of AURKA.5.Study the mechanism of SDCBP promoting ESCC cell proliferationWestern blot was used to detect the expression of p-EGFR,p-PI3K,and p-Akt after knocking down SDCBP,or after inactivation of SDCBP phosphorylation.The binding ability of SDCBP and EGFR was verified by endogenous and exogenous coimmunoprecipitation experiments.Immunofluorescence and flow cytometry were used to detect changes in the membrane localization of EGFR after knockdown of SDCBP,or after inactivation of SDCBP phosphorylation.Patient derived xenograft(PDX)animal model was performed to verify that SDCBP regulated EGFR/PI3K/Akt signaling pathway.Results1.AURKA’s interacting proteins were related to acetylation,RNA binding,phosphorylation,and methylation.After screening and verification,SDCBP may be identified as the downstream substrate protein of AURKA in ESCC.AURKA and SDCBP bind to each other in ESCC cells and 293T cells.AURKA could bind to the PDZ1 domain of SDCBP but not to the PDZ2 domain.The expression of AURKA and SDCBP in ESCC cell lines and patient cancer tissues were positively correlated.2.From in vitro experiments,AURKA directly phosphorylated SDCBP.Based on the the Netphos2.0 website and AURKA phosphorylation consensus motif,the three sites of SDCBP(Ser87,Ser131 and Thr200)may be phosphorylated by AURKA.After Ser87,Ser131 and Thr200 single-site phosphorylation inactivation mutations,the phosphorylation level of SDCBP decreased only after Ser131 and Thr200 single-site phosphorylation inactivation,indicating that SDCBP was phosphorylated by AURKA at Ser131 and Thr200 sites.The phosphorylation of SDCBP decreased more significantly after double inactivation mutation of both Ser131 and Thr200 sites.3.After AURKA knockdown,the expression of SDCBP protein decreased but the mRNA level did not change.After AURKA overexpression,the protein level of SDCBP increased but the mRNA level did not change,indicating that the regulation of SDCBP by AURKA was at the post-transcriptional level.After CHX treatment,knocking down AURKA promoted the degradation of SDCBP,and overexpression of AURKA inhibited the degradation of SDCBP protein.Further research showed that knocking down AURKA promoted the ubiquitination of SDCBP.Inactivation of SDCBP phosphorylation sites promoted the degradation of SDCBP caused by CHX treatment and increased the ubiquitination of SDCBP.4.Expression of SDCBP was increased in patient cancer tissues,and the high expression of SDCBP was related to the advanced clinical stage,indicating a poor prognosis for ESCC patients.ESCC cell proliferation and colony formation ability decreased after SDCBP knockdown,or after SDCBP phosphorylation was inactivated.ESCC cell proliferation and colony formation ability increased after SDCBP overexpression.The CDX model demonstrated that after knocking down SDCBP,the growth of the tumor was significantly slowed down.SDCBP overexpression couldrecover the proliferation inhibition caused by AURKA knockdown,indicating that SDCBP was a functional downstream substrate of AURKA.5.Western blot results indicated that after knocking down SDCBP or inactivation of SDCBP phosphorylation,the expression levels of p-EGFR,p-PI3K,and p-Akt decreased,indicating that the EGFR/PI3K/Akt pathway was inhibited.Endogenous and exogenous co-immunoprecipitation experiments demonstrated that SDCBP and EGFR bind to each other,and EGFR could bind to the PDZ2 domain of SDCBP.Immunofluorescence and flow cytometry analysis assays showed that after knocking down SDCBP,or after inactivation of SDCBP phosphorylation,the expression of cell membrane EGFR decreased.PDX animal model results showed that knocking down SDCBP inhibited ESCC tumor growth through the EGFR/PI3K/Akt pathway.ConclusionsUpregulated SDCBP in ESCC maintains its protein stability,promotes ESCC proliferation and activates EGFR/PI3K/Akt pathway through phosphorylated by AURKA at Ser131 and Thr200 sites.Targeting AURKA-SDCBP-EGFR axis can provide theoretical basis for ESCC treatment.