The Mechanism Of EGFR Degradation Induced By KTN1 Depletion In Cutaneous Squamous Cell Carcinoma (cSCC)

BackgroundKinectin1(KTN1)is a ligand isoform of microtubule protein kinesin,which locates on the endoplasmic reticulum(ER)membrane and is one of the constituent proteins of the endoplasmic reticulum.Multiple variants and splices of KTN1 have been found in human hepatocellular tumor,suggesting the correlation between KTN1 and tumors.Our previous study demonstrated that MALAT1-KTN1-EGFR regulatory axis promotes the development of cutaneous squamous cell carcinoma(cSCC)and found that KTN1 reduction inhibits EGFR protein translation.EGFR is an important member of the epidermal growth factor receptor family and distributed in various cell membrane systems of the human body.The aberrant of EGFR expression plays a crucial regulatory role in downstream signal transduction and is closely related to the tumorigenesis,development and prognosis of various cancer.Studies have shown that EGFR is widely involved in regulating many biological processes of epithelial squamous cell carcinoma,but there is no report on the function of KTN1 in cSCC.Our finding proved the relationship between KTN1 and EGFR signal pathway transduction and its role in cSCC,but how KTN1 regulates the translation process of EGFR has not been elucidated yet.Therefore,it is particularly important to explore the mechanism of KTN1 regulating EGFR in cSCC.Based on the previous result that knockdown of KTN1 reduces the expression of EGFR proteins.It is necessary to confirm whether the result is mediated by protein synthesis or degradation.In addition,iTRAQ was used to screen differential regulatory proteins,and other experiments were used to explore the function of target proteins and the mechanism of KTN1 in regulating EGFR translation.Finally,nude mice xenograft model is used to verify the regulatory mechanism.Methods1 qPCR and western blot were used to detect the expression of EGFR mRNA and protein after siKTN1 and siNC transfection in the A431,HSC-5 and HaCaT cell lines respectively.Western blot was used to detect the expression of KTN1 protein after siEGFR and siNC transfection in the A431 and HSC-5 cell lines respectively.Folw cytometry and colony formation assay were used to detect apoptotic ratio and survival fraction after siKTN1 and siNC transfection in the A431,HSC-5 cell lines respectively.2 MG 132 or CHX were added to siNC or siKTN1 transfected A431 and HSC-5 cell lines,western blot were used to detect the expression of EGFR,ubiquitin and UCH37.MG132 was added to siNC or siKTN1 transfected A431cell line to detect the protease activity.Co-IP was used to detect the interaction of EGFR and ubiquitin.3 iTRAQ was conducted in A431 cell transfected with siNC and siKTN1 to explore differentially expressed protein.qPCR was used to find upregulated subunits of proteasome in A431 and HSC-5 cell lines.4 Western blot was used to detected c-Myc protein level in cSCC cells after knockdown of KTN1.ChIP-qPCR was performed to confirmed which predicted promoter could interact with c-Myc.Luciferase assay was used to detect the transactivity of 2 predicted promoters.EMSA was used to confirm the direct binding of c-Myc and promoter.5 Western blot was used to detected the CCDC40-ADRM1-UCH37 regulatory axis and the change of EGFR expression.6 Western blot and native gel analysis were used to detect expression of PSMA1 after siKTN1 or siNC transfection in A431 and HSC-5 cell lines.Western blot was used to detect expression of EGFR after siKTNl or siNC transfection in A431 and HSC-5 cell lines.Folw cytometry and colony formation assay were used to detect apoptotic ratio and survival fraction after siKTNl and siPSMA1 or siNC and siPSMA1 transfection in the A431,HSC-5 cell lines respectively.Nude mice xenograft model is used to verify the regulatory mechanism.7 Immunofluorescent staining was used to detect localization of KTN1,PSMA1 and ADRM1.Co-IP assay was carried out to ensure the interaction among KTN1,PSMA1 and ADRM1.8 Mouse xenografts model of cSCC cell was performed to prove the function of the EGFR degradation regulatory axis triggered by KTN1.Results1 Knockdown of KTN1 reduces EGFR protein expression via translation processing and increases apoptosis but represses survival fraction of CSCC cells.2 KTN1 regulates EGFR degradation via UPS.3 CCDC40,PSMA1,ADRM1 are upregulated by knockdown of KTN 1.4 c-MYC is upregulated by knockdown KTN1,which directly binds into the promoter region to transactivate CCDC40.5 CCDC40-ADRM1-UCH37 axis plays a crucial role in deubiquitination of EGFR.6 PSMA1 represents a key modulator in KTN1 mediated EGFR degradation in vivo and in vitro.7 KTN1 interacts with PSMA1 and ADRM1 each other,and competitive binds into ADRM1 in amino acid residues Met1-Ala252.8 In vivo function of the EGFR degradation regulatory axis triggered by KTN1.ConclusionKTN1 depletion promotes EGFR degradation via UPS activation in cutaneous squamous cell carcinoma.