CKAP4 Promotes Hepatocellular Carcinoma Progression And ER-phagy Via Competing With TRIM21 To Bind FAM134B

Objective: Hepatocellular carcinoma(HCC)is the fifth malignant tumor and the second cause of death in China.Early intrahepatic metastasis and postoperative recurrence are the main causes of death.Therefore,it is extremely urgent to study the molecular mechanism of HCC proliferation and metastasis to find the effective molecular targets for the treatment of HCC.Cytoskeleton-associated protein 4(CKAP4)has been proven to play a role in tumor progression in a variety of cancers,and anti-CKAP4 polyclonal antibody has a significant effect on the treatment of subcutaneous tumor in mice.The purpose of this study is to explore the biological function and mechanism of CKAP4 in hepatocellular carcinoma.Methods: Immunohistochemistry(IHC)and Western blot were used to verify the expression of CKAP4 in hepatocellular carcinoma and its corresponding non-tumor tissues.The overexpression and knockout cell lines of CKAP4 were constructed,and the effect of CKAP4 in vitro was verified by cell counting kit-8(CCK-8)assay,cloning formation assay,and Transwell experiment.The effect of CKAP4 in vivo was verified by nude mice orthotopic model,subcutaneous tumor model and tail vein lung metastasis model.Mass spectrometry(MS)was used to find CKAP4-binding proteins.The interaction of CKAP4 and Family with sequence similarity 134,member B(FAM134B)was verified by Coimmunoprecipitation(Co-IP)and immunofluorescence(IF).The regulation of CKAP4 on FAM134B was verified by Western blot,protein half-life assay,MG132 treatment experiment,and ubiquitination-based Co-IP experiment.The potential E3 ubiquitin ligase Tripartite motif-containing 21(TRIM21)of FAM134B was screened by mass spectrometry,and the regulation of TRIM21 on FAM134B was verified by Co-IP,IF,Western blot,protein half-life assay,MG132 treatment experiment,and ubiquitination-based Co-IP experiment.Co-IP confirmed that CKAP4 competed with TRIM21 to bind FAM134B.Flow cytometry confirmed that CKAP4 and TRIM21 regulated apoptosis which is related to endoplasmic reticulum(ER)stress by regulating FAM134B.Results: CKAP4 was upregulated in hepatocellular carcinoma,and its high expression was associated with poor prognosis.Overexpression of CKAP4 promoted hepatocellular carcinoma cell proliferation and metastasis in vivo and in vitro,while knockout of CKAP4 inhibited proliferation and metastasis.CKAP4 promoted the development of hepatocellular carcinoma via inhibiting the ubiquitin-proteasome pathway degradation of FAM134B.TRIM21 interacted with FAM134B to increase K48-polyubiquitination on FAM134B K247and K252.CKAP4 and TRIM21 competed to interact with FAM134B to regulate endoplasmic reticulum autophagy(ER-phagy).CKAP4 stabilized FAM134B protein expression,promoted ER-phagy,and resisted apoptosis.TRIM21 increased FAM134B degradation,inhibited ER-phagy,and promoted cell apoptosis.Conclusions: CKAP4 is upregulated in hepatocellular carcinoma and its high expression is associated with poor prognosis.By competing with TRIM21 to bind FAM134B,CKAP4protects FAM134B from ubiquitin-proteasome degradation.CKAP4 promotes the proliferation and metastasis of hepatocellular carcinoma via stabilizing FAM134B.CKAP4and TRIM21 participate in the regulation of ER-phagy by regulating FAM134B.