Targeting FAM134B-mediated Reticulophagy Activates Sorafenib Induced Ferroptosis In Hepatocellular Carcinoma

BackgroundPrimary liver cancer is currently the sixth most common malignant tumor and the third leading cause of death worldwide;its complex etiology,insidious development and high malignant degree seriously threaten the life and health of human.Primary liver cancer mainly includes Hepatocellular carcinoma(HCC)、Intrahepatic cholangio carcinoma(ICC)and cHCC-ICC,which differ greatly in pathogenesis,biological behavior,treatment methods and prognosis.Among which,85%-90%are hepatocellular carcinoma.Patients with HCC first diagnosed have often progressed to the middle and late stages,missing the best treatment time,resulting in limited treatment and still poor prognosis.Several clinical studies have shown that the classical first-line targeted drug sorafenib has some survival benefit for patients with advanced HCC.However,with the use of the drug,only about 30%of HCC patients were found to be clinically effective with sorafenib,and resistance to the drug could occur within 6 months of administration.Therefore,further studies on the anti-cancer mechanism of sorafenib is of great significance for the development of drugs and the improvement of the overall survival of patients.Ferroptosis was proposed by Stockwell et al.in 2012 and has received increasing research and attention in recent years.As a new cell death style,ferroptosis is characterized by the aggregation of iron-dependent lipid peroxides and reactive oxygen species(ROS),and severe lipid peroxidation causes intracellular redox imbalance.A growing number of studies have shown that sorafenib induces hepatocellular carcinoma cell death in the main form of ferroptosis rather than apoptosis as previously thought,thus highlighting the importance of ferroptosis in the treatment of HCC.Various regulatory proteins such as retinoblastoma(Rb)protein,long chain non-coding RNA(LncRNA),and other small molecules have been identified,LncRNA,p53 and p62-Keapl-Nrf2 pathways are involved in the regulation of ferroptosis.Autophagy is an evolutionarily conserved process of self-renewal of intracellular material in eukaryotic organisms.According to the specificity of the degradation substrate,autophagy can be divided into selective and non-selective,among which selective autophagy includes lipophagy,ribosomal autophagy,endoplasmic reticulum autophagy(ER-phagy),mitophagy,peroxisome autophagy,etc.In 2015,Khaminets et al discovered the ER-phagy receptor protein FAM134B,then the research on ER-phagy was gradually carried out.Down-regulation of FAM134B resulted in the expansion of the ER,whereas overexpression of FAM134B promoted the fragmentation of the ER into lysosomes.Selective autophagy was also found to be closely related to ferroptosis,it can regulate the sensitivity of cells to iron death by regulating intracellular iron homeostasis or lysosome dependent lipid peroxidation.However,the relationship between ER-phagy and ferroptosis is still not elucidated.Relying on the previous research results of two National Natural Science Foundation projects of our research team,this project carried out in-depth research on the regulatory mechanism between ER-phagy and ferroptosis in hepatocellular carcinoma cells.In the present study,we found that sorafenib can effectively activate ER-phagy mediated by receptor protein FAM134B,Downregulation of FAM134B not only inhibited endoplasmic reticulum autophagy but also significantly enhanced the sensitivity of HCC cells to ferroptosis without affecting macro autophagy.Similar results were obtained in tumorigenic experiments of nude mice.Subsequently,the results of combined bioinformatics analysis,RNA-binding protein immunoprecipitation and polyribosome isolation experiments tentatively showed that PABPC1 could regulate the translation process of FAM134B expression.Taken together,this study reveals the regulatory role of PABPC1-FAM134B-endoplasmic reticulum autophagy pathway in sorafenib-induced ferr opto sis in hepatocellular carcinoma cells,providing important evidence for new anti-cancer strategies and the development of drugs.Part Ⅰ.Sorafenib induces FAM134B target-mediated hepatoma cell endoplasmic reticulum autophagyPurpose of the studyTo clarify the relationship between sorafenib and FAM134B targe ting-mediated ER-phagy,laying the foundation for subsequent experiments.Research methods1.HepG2 and Huh7 cells were treated with different concentrations of sorafenib for 24h,and the expression changes of endoplasmic reticulum autophagy-targeting protein FAM134B and endoplasmic reticulum in situ protein were detected by Western-blot assay to understand the effect of sorafenib on FAM134B-targeted endoplasmic reticulum autophagy.2.HepG2 and Huh7 cells were treated with sorafenib at 5uM concentration for 24h,and the changes of organelles,autophagic vesicles and lysosomes in the cells were observed under electron microscope to analyze the effect of sorafenib on endoplasmic reticulum autophagy.Study results1.Sorafenib significantly inhibited FAM1 34B targeting-mediated endoplasmic reticulum autophagy.Compared with the control group,the FAM134B expression levels of both hepatocellular carcinoma cell lines in the sorafenib-treated group showed a significant decrease and a negative correlation trend with sorafenib dosing concentration in the 1.25 uM-10 uM concentration range;the electron microscopy results of HepG2 and Huh7 cells with sorafenib treatment for 24h revealed swollen and deformed endoplasmic reticulum,typical autophagic vesicles,lysosome formation,and degraded endoplasmic reticulum fragments,clearly confirming the occurrence of endoplasmic reticulum autophagy.2.Both hepatocellular carcinoma cell lines of endoplasmic reticulum in situ protein Trap-a and REEP5 showed similar trends to FAM134B after sorafenib treatment.ConclusionSorafenib activates FAM134B-mediated endoplasmic reticulum autophagy in hepatocellular carcinoma cells.Part Ⅱ Targeting FAM134B-mediated endoplasmic reticulum autophagy to regulate ferroptosisPurpose of the studyTo investigate the effect of targeting FAM13 4B-mediated endoplasmic reticulum autophagy on sorafenib-induced ferroptosis in hepatocellular carcinoma cells.Research methods1.FAM134B knockdown HepG2 and Huh7 cell lines were constructed using a transient transfection method.2.Application of protein fluorescence co-localization assay to observe the effect of FAM134B knockdown on endoplasmic reticulum autophagy in hepatocellular carcinoma cells after sorafenib treatment.3.Western-blot was applied to detect changes in the expression of FAM134B,autophagy vesicle receptor protein LC3B and endoplasmic reticulum surface protein Trap-a,key regulator of ferroptosis GPX4,after FAM134B knockdown,to investigate the relationship between ER-phagy autophagy and sorafenib-induced ferroptosis and identify whether FAM134B knockdown had an effect on macroautophagy.4.Iron assay kit was applied to detect the iron content and observe the effect of FAM134B knockdown on the iron content in hepatocellular carcinoma cells after sorafenib treatment.5.The ROS assay kit was applied for the detection of ROS content,and the effect of FAM134B knockdown on the ROS content in sorafenib-treated hepatocellular carcinoma cells was observed using confocal microscopy and cell flowmetry.6.Knockdown FAM134B and negative control HepG2 cells were injected subcutaneously for tumorigenic experiments in nude mice,respectively.7.The dose of sorafenib was calculated at 10 mg/kg body weight of nude mice and was administered by intraperitoneal injection,once every 2 days for 4 weeks.8.The tumors were harvested,and the weight and volume of the tumors were measured to observe the effect of FAM134B knockdown on tumor formation in nude mice.9.Tumor tissue proteins were extracted and the expression of FAM134B,LC3B and GPX4 were detected by applying Western-blot to observe the effects of FAM134B knockdown on endoplasmic reticulum autophagy,macroautophagy and ferroptosis.Study results1.The results of protein fluorescence co-localization assay showed that the expression of FAM134B on the endoplasmic reticulum surface of hepatocellular carcinoma cells in both HepG2 and Huh7 groups was significantly reduced after lentivirus transfection with si-FAM134B.2.The results of protein fluorescence co-localization assay showed that the endoplasmic reticulum autophagy receptor FAM134B co-localized significantly with autophagosome surface receptor LC3B after FAM134B knockdown in HepG2 cell line.The same trend was shown in Huh7 cell line.3.Western-blot assay showed that FAM134B expression was significantly reduced after FAM134B knockdown in HepG2 cell line,but did not affect the expression of LC3B.The same trend was shown in Huh7 cell line.4.The results of Western blot assay showed that the GPX4 content of HepG2 cell line decreased in control group and FAM134B knockdown group after the treatment of sorafenib(p<0.01);the GPX4 content of FAM134B knockdown group decreased more significantly after the treatment of sorafenib(p<0.01).The same trend was also observed in Huh7 cell line.5.The results of the iron content assay showed that the iron content of HepG2 cell lines treated with sorafenib increased in the control and FAM134B knockdown groups;the iron content of FAM134B knockdown group of hepatocellular carcinoma cells treated with sorafenib increased more significantly(p<0.01).The same trend was observed in Huh7 cell lines.6.The ROS kit-treated HepG2 cells were observed by confocal microscopy and flow cytometry respectively,and the results showed that the ROS content was significantly higher in the FAM134B knockdown group after sorafenib treatment compared to the control group.The same results were presented in the Huh7 cell line.7.The tumors were harvested and the weight and volume of tumors were measured.And the results showed that the weight and volume of tumors in the FAM134B knockdown group were significantly smaller than those in the control group(p<0.01).8.The results of Western blot showed that the expression of FAM134B and GPX4 were significantly decreased in the tumor tissues of the FAM134B knockdown group compared with the control group,while the expression of LC3B was not affected.Conclusion1.Inhibition of FAM134B-mediated endoplasmic reticulum autophagy did not affect macroautophagy in hepatocellular carcinoma cells.2.Inhibition of FAM134B-mediated endoplasmic reticulum autophagy enhances sorafenib-induced ferroptosis in hepatocellular carcinoma cells without affecting macroautophagy.3.Inhibition of fam134b mediated endoplasmic reticulum autophagy can enhance the anti hepatoma effect of sorafenib in nude mice.Part Ⅲ Primary exploration of upstream regulatory mechanism of FAM134BPurpose of the studyExploring the upstream regulatory proteins of FAM134B to further investigate the signaling pathway.Research methods1.Bioinformatics methods were applied to predict potential binding proteins of FAM134B mRNA by RBPmap and RBPDB information libraries.2.RNA immunoprecipitation experiments were applied to further screen for relatively highly expressed RNA-binding proteins from the predicted results.3.The target protein was down-regulated using lentiviral transfection method and its effect on FAM134B expression was observed by qRT-PCR and Western-blot assays.Study results1.Four potential regulatory proteins,including PABPC1,HuR,SRSF1 and MBNL1,were screened by cross-matching between RBPmap and RBPDB information bases.2.Application of RNA immunoprecipitation experiments revealed the highest RNA content of FAM134B in the anti-PABPC1-mRNA complex,suggesting that PABPC1 binds most to FAM134B mRNA and may be a regulatory proteins of FAM134B.3.The results of qRT-PCR experiments showed that FAM134B mRNA expression was not affected after knocking down PABPC1.4.Western-blot assay results revealed that the protein expression level of FAM134B was significantly inhibited after knocking down PABPC1.5.The results of polyribosome concentration gradient centrifugation experiments showed a leftward shift of FAM134B mRNA multimer kurtosis in the PABPC1 knockdown group compared to the control group.Conclusion1.PABPC1 may act as a potential upstream regulatory protein for the regulation of FAM134B expression.2.PABPC1 may achieve the regulation of FAM134B expression through the regulation of post-translational translation of FAM134B mRNA.