Evolution Of The Conserved CNC-bZIP Family With Disversity Of Its Members Nrf1 And Nrf2 In Regulating The Hepatoma Cell Metabolism

Liver cancer is one of the most common malignant tumors with a serious threat to human life.However,it has a poor prognosis,although liver cancer is caused primarily by infection with hepatitis B and C viruses,contamination by aflatoxin and long-term excessive consumption of alcohol,particularly in China.Unfortunately,all the clinical(including surgical and non-surgical)treatments for liver cancer failed to guarantee a good prognosis for patients and prevent its recurrence.This has always been a difficult,hot topic,allowing scientists to have been committed to exploring mecular mechanism underlying the pathogenesis of liver cancer.As a subgroup of the b ZIP(basic-region leucine zipper,BRLZ)superfamily,all the CNC(cap'n'collar)-b ZIP proteins possess a conserved CNC domain,in addition to other complex domains and conserved motifs near their N-termimus.This faminly consists of p45 Nfe2,Nrf1,Nrf2,Nrf3 and their transcriptional inhibitors Bach1 and Bach2 in vertebrates,and the Caenorhabditis elegans protein Skn-1,in addition to the founding Drosophila melanogaster Cnc protein.They are involved in transcriptional regulation of target genes by forming functional heterodimers with their cognate partners(e.g.,Maf G,Maf K and Maf F)before binding their specific cis-regulatory elements(e.g.,ARE/Ep RE or AP-1)in their promoter regions.Such genes play vital roles in a variety of biological processes,such as antioxidant,cell proliferation and aging,DNA damage repair and cell metabolism.However,almost no study has been focused on the evolution of CNC-b ZIP family genes before herein.Among this family,Nrf1 and Nrf2 have essential antioxidant function,but Nrf1-/-,rather than Nrf2-/-,knockout mice suffer from embryonic lethality,as a result of blocked mesoderm formation or anaemia,implying a large difference between Nrf1 and Nrf2 in embryonic development.Our group also revealed that Nrf1-/-knockout leads to significantly enhanced migration and proliferation of hepatoma cells,and subcutaneous tumorigenicityin nude mice.Conversely,striking repression of Nrf2-/-hepatoma cells was found.Rather,the molecular mechanism leading to this phenotypic difference has not been explored.Thus,this study had done from the perspective of evolution and cancer metabolism,in order to explore the evolution of CNC-b ZIP family genes and determine distinct roles of between Nrf1 and Nrf2 in hepatoma cells,by using a number of bioinformatics and molecular biology methods.The results are as follows:(1)By combination of homology and structure search,a total of 441 b ZIP proteins were identified;They are widely distributed across all the 23 representative species from viruses,bacteria and protozoa to metazoans.Notably,the number of b ZIP transcription factor increases,as species have evolved from simple to complex.(2)Nachs(Nach1-8),a new subclass of CNC-b ZIP family was identified.Among them,Nach3,5,6,and 7 each has a typical transmembrane domain,which is the same as that of human Nrf1 or Nrf3.By contrast,the early CNC-b ZIP homolog Nach1/2 from marine bacteria is incapable of driving ARE reporter gene activity,but they negatively regulates Nrf1/2-mediated ARE reporter activity in Hep G2 cells.(3)Nrf1 plays a central role in the human b ZIP interaction network,and thus the expression of other b ZIP genes are altered by Nrf1?-/-konckout or its induction.(4)Under the normal cultured conditions,Nrf1?-/-knockout promote glycolysis,gluconeogenesis,pentose phosphate pathway,amino acid synthesis and transport,and fatty acid accumulation,as accompaned by reduced mitochondrial oxygen consumption in hepatoma cells.By contrast,knockout of Nrf2-/-inhibits glycolysis,gluconeogenesis,and amino acid synthesis and transport,but no changes in pentose phosphate pathway along with no accumulation of fatty acids.(5)In the glucose-free culture,Nrf1?-/-Hep G2 cells showed rapid non-apoptotic cell death(as observed after glucose starvation for 12 h).By contrast,Nrf2-/-cells has a certain capability to resist the cellular death caused by glucose deprivation(as the cells are growing well after glucose starvation for 24 h).(6)The rapid death of Nrf1?-/-cells caused by glucose starvation was resulted from disrupted antioxidant systems,abnormal glucose and energy metabolism,and blocked serine-to-glutathione synthesis pathways.Altogether,these results indicate that the early CNC-b ZIP homolog Nach1/2 exists in marine bacteria,acting as negative regulators of human Nrf1/2.Such distinct roles of between Nrf1 and Nrf2 in metabolic reprogramming of hepatoma cells are embodied by the facts that Nrf1?-/-cells are extremely sensitive to glucose deprivation,as compared with Nrf2-/-cells.Thereby,glucose starvation is much likely to become an effective strategy for the prevention and treatment of Nrf1-deficient cancer.