CD47 Regulates Redox Status By MTORC1 And Promotes Hepatocellular Carcinoma Growth

Background&Aims:Liver cancer is the second most common cause of cancer-related death in the world.The major form of primary liver cancer is hepatocellular carcinoma?HCC?.Seeking new molecules for early diagnosis and therapy of HCC is urgent.CD47 is a quantic-transmembrane glycoprotein that is ubiquitously expressed in normal tissues and mediates a‘don't eat me'signal on normal cells by inhibiting macrophage phagocytosis through its interaction with macrophage signal regulatory protein alpha?SIRP??.The important pathophysiological functions of CD47 in cardiovascular homeostasis,immune regulation,resistance of cells and tissues to stress and chronic diseases including cancer have been revealed.The enhanced expression of CD47 has also been reported in various malignancies including leukemia,lymphoma,multiple myeloma and solid tumors such as breast,colon,melanoma and hepatocellular carcinoma.CD47 is preferentially expressed in HCC tumor-initiating cells which contributes to tumor initiation,self-renewal and metastasis,and significantly affects the clinical outcome of patients.NF-?B mediating CD47 upregulation promotes sorafenib resistance,and targeting CD47 in combination with sorafenib is an attractive therapeutic regimen for the treatment of HCC patients.The mTOR protein is a ubiquitously expressed serine–threonine protein kinase,and a member of the PI3K-related kinase family.It acts as a cellular energy sensor by integrating signals from hormones,energy levels,growth factors,and nutrients into the regulation of protein translation,autophagy,and lipid metabolism.The ability of mTOR to regulate cell function is achieved through the existence of two structurally and functionally distinct complexes,mTORC1 and mTORC2.mTORC1 comprises mTOR,RAPTOR,mLST8,PRAS40,and DEPTOR.The mTORC1 complex is activated by amino acids,growth factors and nutrients,and its activation is associated with augmented cell survival because it results in increased protein synthesis,initiation of lipid biosynthesis,and diminished autophagy.Moreover,mTORC1 can increase Nrf2 protein levels.Nuclear factor?erythroid-derived 2?-like 2,also known as NFE2L2 or Nrf2,is a transcription factor that regulates the expression of antioxidant proteins that protect against oxidative stress.mTORC1 can stabilize Nrf2 by phosphorylating Ser-351 in p62/SQSTM1 and thereby enabling p62/SQSTM1 to bind to Keap1 to diminish Keap1 mediating degradation of Nrf2.In our study,we found CD47 could promote HCC growth and help HCC cells to resist to oxidative stress by regulation of mTORC1.Our work aimed to explore the non-immunological pathophysiological functions of CD47 in HCC in order to get a more thorough understanding of its function and to explore the relevant mechanisms.Our study hoped to lay a foundation for CD47 as a target for diagnosis and treatment of HCC.Methods:1.We used a lentiviral-based approach to establish HCC cells with different CD47expression levels,including CD47 overexpressed,knockdown and control cells.2.We adopted CCK-8 to measure the proliferation of cells with different CD47expression in two-dimension-culture conditions.Soft agar,Matrigel and ultra-low-attachment sphere formation assays were conducted to explore the growth of cells with different CD47 expression in three-dimension-culture conditions.3.Western blot analysis was employed to detect the alteration of proteins and their phosphorylation in related signaling transduction pathways under multiple treatment conditions.4.Real-time PCR technology was used to analyze antioxidant-related genes and other genes expression.5.We used flow cytometry to analyze the expression levels of CD47 and SLC3A2.Combined with reactive oxygen probe flow cytometry was employed to measure the ROS levels in different conditions and with Annexin V/PI apoptosis detection kit to detect apoptosis induced by H₂O₂.6.We conducted amino acid mass spectrometry,which showed the fluctuations of amino acid content.We identified proteins interacting with CD47 by coimmunoprecipitation assay combined with silver staining and LC-MS/MS.7.Cellular immunofluorescence technique was adopted to analyze intracellular ROS levels and the co-location of CD47,SLC3A2 and lipid rafts.Tissue immunofluorescence technique was used to analyze the co-location of CD47 with SLC3A2 or p-p38 in HCC specimen.8.The coimmunoprecipitation assay was applied to confirm CD47 and SLC3A2interactions and to analyze the ubiquitination status of Nrf2 and SLC3A2.9.Crystal violet staining combined with Microplate Spectrophotometer absorption detection in 590 nm was used to analyze drug sensitivity.10.We used MoS₂ to synthesize 2D biocomposites as molecular probe to detect CD47expression in HCC specimen.Results and conclusion:1.To study the effect of CD47 on mTORC1 signaling pathway in HCC cells:CD47promoted the 3D growth of HCC cells in a mTORC1 dependent way.CD47 regulated mTORC1 activity by influencing intracellular amino acid content.2.To study the regulation of redox status in HCC cells by CD47 through mTORC1:CD47 stabilized Nrf2 to regulate redox status in HCC cells by modulating mTORC1.CD47boosted oxidative stress resistance ability in HCC cells.3.To study the mechanism of CD47 influencing mTORC1:CD47 interacted with SLC3A2 and enhances its stability.CD47 regulated mTORC1 signaling pathway in HCC cells through SLC3A2.4.To explore the diagnosis and treatment of HCC by targeting CD47:CD47 antibody could elevate ROS to increase the sensitivity of HCC cells to oxaliplatin.Fluorogenic 2D biocomposite unraveled the high expression of CD47 in HCC tissues.Our results revealed the function of CD47 that contributed to the mTORC1 pathway activation and the oxidative stress resistance in HCC cells.CD47 in HCC cells could interact with SLC3A2 to stable its expression in cell membrane and then influence intracellular amino acid content.Leucine?Leu?,tryptophan?Trp?and phenylalanine?Phe?showed an intracellular content increase when CD47 was overexpressed,which could activate mTORC1 signaling pathway.mTORC1 activation resulted in the elevation of the phosphorylation of p62,causing the stabilization of Nrf2,which then induced cytoprotective target genes?Fig 15?.More importantly,CD47 antibodies?clone 2D3,BRIC126 and CC2C6?could elevate cell ROS levels and improve chemosensitivity.We also synthesized 2D biocomposite as CD47 molecular probe and provided data for CD47 as a target for diagnosis and treatment of HCC.