Roles Of SLC47A1-Mediated Lipid Remodeling In Cancer During Ferroptosis

Objectives: To screen and identify molecules within the phospholipid translocation enzyme family involved in regulating ferroptosis in cancer cells;to investigate the role and mechanism of the phospholipid translocation enzyme SLC47A1 in regulating ferroptosis in cancer cells;to assess the synergistic anti-tumor effects of inhibiting SLC47A1 and inducing ferroptosis.Methods: We constructed a pancreatic cancer ferroptosis model,and using real-time fluorescence quantitative polymerase chain reaction(q RT-PCR),we screened for differentially expressed phospholipid transporters during the ferroptosis process,and conducted a protein immunoblotting(WB)experiment to detect the expression level of the phospholipid transporter SLC47A1 in the ferroptosis model.We then constructed a si RNA library for phospholipid transporters to screen for key molecules regulating ferroptosis.Using short hairpin RNA(sh RNA)or overexpression plasmid lentivirus,we constructed stable SLC47A1low-expression and overexpression cell lines in various tumor models,including leukemia,pancreatic cancer,and lung cancer.To verify the function of SLC47A1 in cancer during ferroptosis,we used CCK-8experiment,lipid ROS detection and MDA detection.The mechanism of SLC47A1 regulating ferroptosis in cancers was explored using liquid chromatography-tandem mass spectrometry(LC-MS/MS)and molecular docking model analysis methods.The mechanism was further verified by using CRISPR-Cas9 technology to construct stable SLC47A1,ACLS4,and SOAT1 knockout cell lines.Finally,we explored the upregulation mechanism of SLC47A1 in ferroptosis using bioinformatics analysis,WB experiment,and CCK-8 experiment.Nude mouse subcutaneous transplantation tumor models of pancreatic cancer,lung cancer,and other cancers were constructed to evaluate the value of gene or drug inhibition of SLC47A1 and inducing ferroptosis in synergistically exerting anti-tumor effects.Results:(1)In various cancer ferroptosis models induced by RSL3 or Erastin,the phospholipid flippase SLC47A1 was significantly upregulated.Moreover,the expression level of SLC47A1 increased in a time-and concentration-dependent manner with the occurrence of ferroptosis.This upregulation of SLC47A1 was reversed by the ferroptosis inhibitors,ferrostatin-1 and liproxstatin-1(2)Through screening of a phospholipid translocation enzyme si RNA library,it was found that interference with the expression of SLC47A1 significantly promoted ferroptosis induced by RSL3 or Erastin.Furthermore,it has been confirmed in various cancer cell lines,such as acute leukemia,pancreatic cancer,and lung cancer,that silencing SLC47A1 promotes ferroptosis,while also promoting the generation of lipid ROS and MDA.Conversely,overexpression of SLC47A1 inhibited ferroptosis occurrence,and these phenotypes could be reversed by ferrostatin-1 or liproxstatin-1.(3)Silencing SLC47A1 leads to the accumulation of a large amount of lipids in cancer cells,among which the increase in polyunsaturated fatty acid-containing cholesterol esters(PUFA-CE)is the most significant(VIP>1,P<0.05).SLC47A1 may be associated with the flipping of polyunsaturated fatty acids DHA and DPA,and silencing SLC47A1 promotes the accumulation of DHA and DPA,thus increasing the content of intracellular PUFA-CE.In cell lines with overexpression of SLC47A1,the exogenous supplementation of DHA or DPA significantly sensitizes ferroptosis.(4)Knocking out the fatty acid activation enzyme ACSL4 or cholesterol ester synthesis enzyme SOAT1 can reverse the increased sensitivity to ferroptosis caused by SLC47A1 deficiency,while also inhibiting the generation of lipid ROS.(5)Transcription factor PPARA positively regulates the expression of SLC47A1 in ferroptosis.Silencing PPARA significantly reduces the expression of SLC47A1 and promotes the occurrence of ferroptosis in tumor cells,while promoting the generation of lipid ROS.(6)Silencing PPARA or SLC47A1 can increase the sensitivity of various transplant tumors to ferroptosis inducers.The SLC47A1 inhibitor cimetidine has a synergistic anti-tumor effect with the ferroptosis inducer IKE.Conclusions:(1)Phospholipid flippase SLC47A1 is a key molecule negatively regulating ferroptosis in cancer cells,whose expression is transcriptionally regulated by PPARA.(2)SLC47A1 mediates cholesterol ester metabolism reprogramming via the ACSL4-SOAT1 axis,thereby inhibiting ferroptosis in cancer.(3)The SLC47A1 inhibitor cimetidine exhibits synergistic anti-tumor effects with the ferroptosis inducer IKE,and can be a potential sensitizer for inducing ferroptosis in cancer.