NUPR1/AHR/CYP450 Axis Mediates Oxidative Stress And Confers Radiation Resistance Of Hepatocellular Carcinoma

Background:According to GLOBOCAN 2020,primary liver cancer is predicted to be the sixth most commonly diagnosed cancer and the third leading cause of cancer death worldwide,which includes 80%of hepatocellular carcinoma(HCC).Poor response to therapies contributes to dismal prognosis of this refractory disease.Radiotherapy(RT)is one of the few therapies with demonstrated clinical feasibility for patients with HCC but provides limited survival benefits due to intrinsic or acquired radiation resistance.Accumulating studies have shown that cells can be damaged indirectly by radiationinduced reactive oxygen species(ROS),which are generated from ionization of water or leakage of electrons in the mitochondrial respiratory chain.Oxidative stress caused by excessive ROS significantly affects the efficacy of radiotherapy.NUPR1 is known as regulating the stress response stimulated by external environment and the homeostasis in body.Under a variety of stress conditions,it mediates multiple biological processes such as autophagy,apoptosis,DNA damage repair,and ferroptosis.However,the mechanism of NUPR1 regulating oxidative stress and mediating the radiation resistance of HCC is unclear.Therefore,using a series of experiments,we firstly explored the functional role of NUPR1 in regulating radiation resistance of HCC.Secondly,we revealed the molecular mechanism of NUPR1,that promotes HCC cells to resist radiation by inhibiting CYP450 expression and restraining oxidative stress.Finally,we clarified the possibillity of specific inhibition of NUPR1 for sensitization of radiotherapy in HCC.Methods:1.HCC Cells were transfected with lentivirus to stably over-express or knockdown NUPR1’s expression.Colony formation assays with increasing doses of radiation(OGy,2Gy,4Gy,6Gy,8Gy)were performed to HCC cells with different NUPR1 status.Comet assay,pH2AX measurement and subcutaneous xenograft tumor model were used to determine the functional role of NUPR1 on radioresistance of HCC.2.Using Transcriptome sequencing,western blot,qRT-PCR determined the effect of inhibition on AHR and CYP450 enzymes expression while over-expressed NUPR1.Cells were culture with fluorescence probe such as H2DCFDA or BODIPY and then decteced by flow cytometry to examine ROS levels and lipid peroxidation,which revealed NUPR1 is able to repress oxidative stress in HCC cells under radiation.3.A series of experiments including western blot,qRT-PCR,immunoprecipitation,immunofluorescence staining,colony famation assays,ROS and apoptosis detection were used to reveal the molecular mechanism of NUPR1/AHR/CYP450 signal axis in regulating radiation resistance of HCC cells.4.Through public database analysis,detection of paraffin-embedded sections by immunohistochemistry and so on,we try to explore the correlation between the expression of NUPR1 and radiatherapy effect and predict prognosis of HCC patients,and to clarify the possibility of targeted inhibition of NUPR1 to enhance radiosensitivity of HCC.Results:1.Over-expression or knock-down of NUPR1 can promote or inhibit the radiation resistance of HCC cells.2.NUPR1 down-regulates the expression of CYP450 and inhibits oxidative stress,thereby promotes radiation resistance of HCC cells.3.NUPR1 negatively regulates the transcription of CYP450 by promoting lysosome degradation of AHR,which is a key mechanism for HCC cells to maintain redox homeostasis under radiation.4.NUPR1 was significantly high expressed and AHR was significantly low expressed in radiation resistant HCC tissues by immunohistochemistry.5.Public database analysis showed that HCC tissues with high expression of NUPR1 and low expression of AHR,CYP1B1 or CYP3A4 can predict the poor prognosis of HCC patients.Conclusions:In this study,we confirmed that NUPR1 binds to AHR and promotes the degradation of AHR by lysosome,thereby down-regulates the transcriptional expression of CYP450,reduces the oxidative damage under radiation,which confers the radiation resistance of HCC cells.Further more,knockdown or specific inhibition of NUPR1 can aggravate oxidative stress and enhance the radiosensitivity of HCC cells.This study reveals a new mechanism of NUPR1 promoting radiation resistance of HCC and provides new therapeutic strategies for sensitization of radiotherapy in HCC.