The Function And Molecular Mechanism Of Redox-sensitive Modulator—Pirin In Ferroptotic Cancer Cell Death

Ferroptosis is a newly defined cell death type in recent years.It is recognized as an iron-dependent,lipid peroxidation induced non-apoptotic cell death.The major cytological changes of cells in ferroptosis include cell volume shrinkage,enhanced mitochondrial membrane density,and does not display the typical characteristics of apoptosis or necrosis.In cancer development,One of the hallmarks of cancer cells is the ability to evade the programmed mechanisms of cell death,which becomes the reason of drug resistance.Comparing to the normal cellular process,high amount of iron are needed in cancer cell proliferation which causes a high sensitivity to ferroptosis.Therefore,inducing ferroptosis in the cancer cells has become one of the important directions of cancer treatment.The classic ferroptotic stimuli has shown the effectiveness and variable sensitivity to induce cell death in different type of cancer cells.It also described as a target therapy for RAS-mutant tumors clinically.Thus,understanding the molecular mechanisms of ferroptosis in cancer cells will provide insights into ferroptosis targeted therapy.The classic ferroptotic stimuli are drugs targeting antioxidant systems,especially the solute carrier family 7 member 11(SLC7A11)-glutathione peroxidase 4(GPX4)axis.Although the process of ferroptosis has cell specificity,iron accumulation and lipid peroxidation are the two most important biochemical events to drive ferroptosis.Organelles are involved in many cellular functions,including cell death.Mitochondria,the endoplasmic reticulum,and lysosomes are the most studied organelles for regulating ferroptosis through metabolic remodeling,protein folding,or autophagic degradation.Other organelles,such as the Golgi apparatus and peroxisomes,also control ferroptosis sensitivity through redox modification or lipid biosynthesis.Although the complex network of organelles decides the occurrence of ferroptosis,molecular communication with organelles is still poorly understood.Pirin is an iron-dependent oxidative stress sensing protein located in the nucleus.It has the function of transcriptional regulation.The expression level of Pirin is relatively low in normal human tissues,however,it is highly expressed in a variety of epithelial-derived cancers.Since Pirin is associated with oxidative stress in cells,we hypothesized that Pirin may play an important role in the lipid peroxidation-related ferroptosis process.In the meanwhile,ferroptosis was reported as a cell death pathway that highly associated with autophagy.We believe that the regulation of Pirin in ferroptosis may be related to the activation of autophagy.In this study,we provide the first evidence that pirin(PIR),an iron-binding nuclear protein,is a key regulator of nuclear stress during ferroptosis.The upregulation of PIR by nuclear factor,erythroid 2-like 2(NFE2L2/NRF2)limits DNA damage during ferroptosis.In contrast,the genetic depletion of PIR increases DNA damage,leading to cytoplasmic translocation and the release of high-mobility group box 1(HMGB1),a DNA chaperone and danger signal,to activate autophagydependent ferroptosis.Finally,blocking the PIR pathway can enhance tumor suppression mediated by ferroptosis in mouse models.These findings establish a key nuclear pathway to regulate ferroptosis.Methods:Four cell lines(PANC1,MIAPa Ca2,Hep G2,A549)were used in the in vitro study.PANC1 was mainly used to investigate the molecular mechanisms.(1)To investigate the expression of Pirin and lipid ROS changes under ferroptosis,PANC1 and MIAPa Ca2 cells were treated with 10 μM Erastin or 0.5 μM RSL3 for 12 hours.To test the function of lipid peroxidation in regulation of Pirin expression under ferroptosis,PANC1 and MIAPa Ca2 cells were cotreated with200 n M Lip-1 or 1m M NAC.q PCR was used to detect Pirin expression at m RNA level and the lipid ROS production was calculated by the shift from 590 nm to 510 nm.(2)To investigate the regulatory function of NFE2L2 on Pirin under ferroptosis,PANC1 and MIAPa Ca2 cells were treated with 10 μM Erastin or 0.5 μM RSL3 together with NFE2L2 inhibitor(ML3855)at 5μM or 10 μM for 12 hours.q PCR was used to detect Pirin expression at m RNA level.sh RNA was used to knockdown endogenous NFE2L2.The protein levels of Pirin and NFE2L2 were detected by using Western Blot and normalized by the level of ACTB.(3)To investigate the role of Pirin on cell death,the endogenous Pirin was knocked down by sh RNA.The cell variability and lipid ROS production was tested in the following groups: Negative control,Erastin 10 μM,Erastin 10 μM + liproxstatin-1200 n M,Erastin 10 μM+ triacsin C 10 μM,Erastin 10 μM+ Z-VAD-FMK 10 μM,RSL3 0.5 μM,RSL3 0.5 μM+ liproxstatin-1 200 n M,RSL3 0.5 μM+ triacsin C 10μM,RSL3 0.5 μM+ Z-VAD-FMK 10 μM.(4)To investigate the regulatory role of Pirin on ACSL4,the endogenous Pirin was knocked down by sh RNA and the cells were treated with 10 μM Erastin or 0.5μM RSL3 for 12 hours.q PCR was used to detect ACSL4 expression at m RNA level.ELISA was used to test the level of arachidonic acid in the culture medium.(5)To investigate the regulatory role of Pirin on DNA damage,the endogenous Pirin was knocked down by sh RNA and the cells were treated with 10 μM Erastin or0.5 μM RSL3 for 12 hours.ELISA was used to test the level of DNA damage markers,γ-H2 AX and 8-oxod G.(6)To investigate the role of Pirin on autophagy pathway,the endogenous Pirin was knocked down by sh RNA and the cells were treated with 10 μM Erastin or 0.5μM RSL3 for 12 hours.The cytoplasmic protein expression of HMGB1,MAP1LC3B-I and-II expression were detected by using Western Blot.The interaction between HMGB1 and BECN1 was detected by using coimmunoprecipitation.ELISA was used to test the HMGB1 in the culture medium.The cell variability was tested in cells with treatment of glycyrrhizin.The in vivo study was conducted in athymic nude mice and evaluated through xenograft model.To evaluate the effects of PIR on ferroptosis,the endogenous Pirin was knocked down by sh RNA and the cells were implanted subcutaneously into the right flank of immunodeficient mice.IKE,a derivative of erastin,was used to treat mice.The tumor volume was recorded.q PCR was used to detect ACSL4 expression at m RNA level in the tumor.ELISA was used to test the MDA and caspase-3 levels in the tumor and HMGB1 level in the blood.Results:1.NFE2L2 mediates PIR upregulation during ferroptosis2.PIR limits ACSL4-dependent ferroptosis3.PIR inhibits ferroptosis by blocking HMGB1-mediated autophagy4.PIR limits ferroptosis-mediated tumor suppression in vivo Conclusions:In summary,we report a new function of PIR in blocking ferroptosis by the modulation of DNA damage-induced autophagic cell death.Targeting PIR signaling can enhance the anticancer effect of ferroptosis therapy in vitro and in vivo.Cancer cells with high epithelial-mesenchymal transition(EMT)status appear to be more sensitive to ferroptosis.Because PIR also contributes to EMT,further research is needed to evaluate whether PIR-mediated EMT is a feedback mechanism for regulating ferroptosis.