Degradation Of ATAD2 To Inhibit The Proliferation And Migration Of Hepatocellular Carcinoma Cells With PROTACs Technology

Background Primary liver cancer,mainly hepatocellular carcinoma(HCC),has a complex pathogenesis mechanism and a high degree of malignancy.Its cancer-related mortality has ranked the second,and the 5-year survival rate is only 18%.Mutations in oncogenes and the pathological environment inducing the formation of cancer lead to the occurrence of liver cancer.Changes in related signaling molecules such as high expression of cancer-associated proteins promote the further development of liver cancer.ANCCA(AAA+nuclear coregulatory cancer-associated protein,also known as ATPase family AAA domain containing 2 or ATAD2)plays an important role in the occurrence and development of almost all cancers as a coactivator of various transcription factors.Moreover,studies have shown that ATAD2 is highly expressed in HCC,which is related to the stage,metastasis,drug resistance,and recurrence of HCC.Although the molecular mechanism of ATAD2-induced HCC is still unclear,inhibition of ATAD2 expression by gene silencing or knockdown can reduce the invasion and proliferation of HCC cells.Recently emerging novel therapeutic strategies use targeted protein degradation(TPD)technology to select different protein degradation pathways to promote the degradation of cancer-related proteins.Proteolysis Targeting Chimeras(PROTACs)is one of the first TPD technologies developed.The artificially designed PROTACs molecule uses three components of E3 ubiquitin ligase-binding ligand,linker and oncoprotein binding ligand to form a hybrid structure,in order to hijack the proteasome pathway mediated by E3 ubiquitin ligase to degradation of oncoprotein.Objective This project intends to use PROTACs technology to design and synthesize fusion peptides PA1 and PA2,specifically degrade ATAD2 protein in liver cancer cells,and then inhibit the signaling pathway related to ATAD2,inhibit the proliferation and migration of liver cancer cells,and achieve the purpose of HCC treatment.Methods(1)PROTACs fusion peptides PA1 and PA2 were designed and synthesized,and their membrane penetrating ability and structures were predicted by bioinformatics.(2)The membrane penetrating effect of fusion peptides in hepatoma cell line Huh7 cells was observed by fluorescence microscope,and the membrane penetrating efficiency of fusion peptides in Huh7 cells was quantitatively analyzed by fluorescence Multiscan Spectrum.(3)Western Blotting was used to detect the degradation of ATAD2 protein in Huh7 cells after fusion peptides treatment and MG132,a proteasome inhibitor,pretreatment.The degradation of BRD2 and BRD4 proteins containing BRD domains in Huh7 cells after treatment with fusion peptides was detected by Western Blotting assays.(4)Detecting the expression of cancer-related protein c-MYC by Western Blotting in Huh7 cells after fusion peptides treatment.(5)CCK8 assay was used to test the effect of fusion peptides on the viability and proliferation of Huh7 cells.(6)The effect of fusion peptides on Huh7 cell migration was detected by Wound Healing assays.(7)TUNEL assay was used to detect the effect of fusion peptides on apoptosis of Huh7 cells.Results(1)PROTACs fusion peptides PA1 and PA2,negative control peptide SS and membrane penetrating control peptides NCOP and R9 HC were synthesized.Bioinformatics analysis showed that the synthesized fusion peptides PA1 and PA2 could penetrate the membrane and their secondary structure is mainly α-helix and random coil.(2)Compared with the membrane penetrating control peptides,fusion peptides PA1 and PA2 and negative control peptide SS had better membrane penetrating ability in Huh7 cells.(3)Fusion peptides PA1 and PA2 could effectively degrade ATAD2 protein in Huh7 cells through the proteasome pathway compared with the negative control peptide SS,PA1 and PA2 could also degrade BRD2 and BRD4 proteins.(4)Compared with the negative control group,fusion peptides PA1 and PA2 could decrease the expression of c-MYC in Huh7 cells.(5)The proliferation of Huh7 cells treated with fusion peptides PA1 and PA2 suppressed significantly within 72 hours compared with the negative control peptide.(6)The crawling speed of Huh7 cells treated with fusion peptides PA1 and PA2 compared with peptide SS was significantly decreased,and the migration of Huh7 cells was inhibited.(7)Huh7 cells treated with fusion peptides PA1 and PA2 not peptide SS can observe obvious fragmentated DNA labelled green fluorescence,and have a shrinking and bright nuclear morphology,which promotes the apoptosis of Huh7 cells.Conclusion PROTACs fusion peptides PA1 and PA2 can effectively penetrate the membrane and degrade ATAD2 protein in Huh7 cells.c-MYC is a well-known cancer-related protein regulated by ATAD2 to promote cancer cell proliferation and inhibit cancer cell apoptosis.It can effectively reduce the expression of c-MYC in Huh7 cells by degrading ATAD2 protein,thereby inhibiting the proliferation and migration of Huh7 cells,promoting the apoptosis of Huh7 cells,and playing an anti-liver cancer effect.This suggests that PROTACs fusion peptides PA1 and PA2 based on CPP could be a potential therapeutic approach in treatment of liver cancer.