Sorafenib Induces Ferroptosis Through ACSL4-mediated Lipid Peroxidation In Liver Cancer Cells

OBJECTIVE Sorafenib is the first-line treatment of advanced hepatocellular carcinoma（HCC）,but it can only prolong the overall survival of advanced HCC patients by 2-3 months.Some HCC patients even obtained acquired resistance to sorafenib.Therefore,it is of great significance to find biomarkers of sorafenib in the treatment of HCC and improve the personalized and precise treatment for HCC patients.In mechanism,apart from the well-established anti-angiogenesis effect,sorafenib can enhance lipid peroxidation and induce ferroptosis,while acyl-Co A synthetase long-chain family member 4（ACSL4）engages in the process of lipid metabolism in cells and promotes ferroptosis.The aim of this study was to explore the role of ACSL4 in the anticancer effect of sorafenib and its predictive effect based upon its expression in HCC tissues.METHODSFirstly,the correlation between the expression of ACSL4 protein and the sensitivity of HCC cells to sorafenib was studied in multiple HCC cell lines in vitro.Secondly,the hypothesis was further verified in nude mice xenograft studies in vivo.Finally,the present study examined the expression of ACSL4 protein in the clinical primary HCC samples and whether ACSL4 expression could be used to predict the prognosis of HCC patients who were treated with sorafenib as adjunct therapy after HCC resection.1.in vitro cellular study:（1）Flow cytometry was used to quantify the protective effects of different cell death inhibitors（e.g.Z-VAD-FMK,Necrostatin-1,Liproxstatin-1）on cell death caused by sorafenib in HCC cells.（2）Western blotting was used to detect the expression of ACSLs in different HCC cell lines.（3）The correlation between the expression of ACSL4 and IC₅₀ of sorafenib was analyzed by Spearman’s rank correlation analysis.（4）Western blotting was used to determine the changes of ACSL4 expression in different HCC cell lines after sorafenib treatment.（5）Flow cytometry was used to quantify the protective effects of ACSL4-specific inhibitors（such as rosiglitazone,pioglitazone）on sorafenib-treated Huh7 cells.（6）Silencing of ACSL4 by si RNA or sg RNA in Huh7 cells to determine the role of ACSL4 in sorafenib-induced ferroptosis.（7）Flow cytometry was used to determine the toxicity of ferroptotic inducers（e.g.erastin,RSL3,BSO）on ACSL4-silienced Huh7 cells.（8）Using BODIPY^TM 581/591 C11,CM-H2DCFDA,GSH assay and MDA assay to detect the lipid peroxidation in Huh7 cells and ACSL4-silenced cells treated with or without sorafenib.2.in vivo xenograft mouse study:Thirty-two male nude mice（aged 6-8weeks）were inoculated with negative control or ACSL4-silenced Huh7 cells,and then randomly divided into four groups:（1）negative control+PBS treatment group;（2）negative control+sorafenib treatment group;（3）ACSL4-silenced cells+PBS treatment group;and（4）ACSL4-silenced cells+sorafenib treatment group.Upon the tumor nodules grow to 20-40 mm³,the mice were given with sorafenib（30mg/kg,every other day,orally）or PBS（same volume）by gavage every other day.The sizes of tumor nodules were measured everyday with calipers and volumes were calculated using the formula（Length×Width×Width）/2.After two weeks’treatment,the mice were sacrificed and the tumor samples were collected.Several indicators of tumor including MDA content were measured,in order to determine the role of ACSL4 in sorafenib treated HCC xenograft in vivo.3.Clinical studies:（1）From the HCC cohort of the First Affiliated Hospital of Guangxi Medical University,clinical information and samples of primary HCC were collected.Inclusion criteria were:ⅰ)the primary treatment was surgery,and then flowed by a 3-month course of sorafenib adjunct treatment;ⅱ)tissue wax blocks after surgery were available;ⅲ)imaging evaluations were availalbe after sorafenib treatment,which can be scaled according to Response Evaluation Criteria in Solid Tumors（RECIST）Version 1.1.A total of 29 patients were enrolled.（2）The expression of ACSl4 protein was detected by Western blotting.（3）The paraffin sections of HCC tissues and adjacent normal liver tissues were collected according to the inclusion criteria.The expressions of ACSL4 in the samples were detected by immunohistochemistry.（4）The relationship between the expression of ACSL4 in tumor tissues and the prognosis of patients was analyzed by Fisher exact test analysis.RESULTS（1）Ferroptotic inhibitors（e.g.Liproxstein-1,Ferrostatin-1,Deferoxamine and GSH-MEE）,rather than pan-Casepase inhibitor（Z-VAD-FMK）or necrosis inhibitor（Nercrostain-1）,protected HCC cells from cell death induced by sorafenib（from 51.77±4.20%to 22.75±0.67%,27.16±0.28%,23.68±1.95%,24.45±1.05%respectively,P<0.05）.（2）The expression of ACSL4 protein was negatively correlated with the IC₅₀of sorafenib in different HCC cell lines（r=-0.952,p<0.001）.（3）In the ACSL4-silenced cells with si RNA（ACSL4-si1and ACSL4-si2）,the sorafenib-induced cell death was less compared with that in the cells with negative control si RNA（22.61±3.76%,25.28±5.59%vs.53.00±4.00%）;Consistently,the stable ACSL4-knockout cells（ACSL4-sg1and ACSL4-sg2）were more resistant to sorafenib than the control stable cells（24.61±0.78%,30.78%±6.12%vs.50.09±1.34%,all P<0.05）.Treatment of ACSL4 inhibitors（rosiglitazone,pioglitazone and 2,4-TZD）decreased sorafenib induced cell death（reduction from 57.00±1.33%to 34.34±4.49%,34.43±1.93%,40.63±1.73%respectively,all P<0.05）.The above results collectively suggested that ACSL4was essential for sorafenib induced ferroptosis.（4）The stable Huh7 cells knocked-out with ACSL4 were more tolerant to cell death induced by different ferroptotic inducers（erastin,RSL3 and BSO）.（5）The lipid peroxidation level of ACSL4-silenced Huh7 cells was lower than negative control Huh7 cells after sorafenib treatment.（6）In the nude mice xenograft studies,sorafenib caused retarded xenograft growth in the negative control group.In contrast,the ACSL4-knockout nodules were resistant to sorafenib treatment and the tumor sizes were almost as same as those without sorafenib.Furthermore,less cell death were observed in the ACSL4-knockout nodules.The Ki-67 immunohistochemistry staining results showed that ACSL4 knockout abolished the inhibitory effects of sorafenib on tumor cell proliferation.（7）ACSL4 was up-regulated in HCC tumor tissues compared with adjacent normal tissues（P=0.000118）.The HCC patients with high expression of ACSL4in their primary HCC tissues had better prognosis after adjunct sorafenib treatment than those with low ACSL4 expression（P=0.029）.CONCLUSIONS（1）Sorafenib induced ferroptosis in HCC cells.（2）Inhibition of ACSL4 rendered Huh7 cells resistance to sorafenib induced ferroptosis reduction of lipid peroxidation.（3）High expression of ACSL4 in HCC primary tumor tissue may predict good prognosis for the post-surgery adjunct sorafenib treatment.