NQO1/PKLR Axis Regulates The Progression Of Breast Cancer By Modulating Glycolysis Reprogramming

Background:Breast cancer(BC)is a common malignancy and a significant cause of death among females worldwide.It is a heterogeneous disease often characterized by its hormone receptor status and expression of human epidermal growth factor receptor 2(HER2).These molecular markers used to classify BC subtypes can also typically predict response to targeted therapies.To date,a number of therapies have been developed to specifically inhibit crucial oncogenic targets in a variety of BC,such as the selective estrogen receptor(ER)modulator,tamoxifen,antagonize ER in estrogen receptor-positive(ER+)cancers and the humanized monoclonal antibody,trastuzumab,inhibits receptor signaling in HER2-amplified/overexpressing cancers.Each therapy achieves a relatively high response rate in their respective patient populations,but invasion and metastasis are still the primary factor that results in the death of BC patients.Therefore,it is urgently needed to find safe and efficient cancer metastasis preventive agents for cancer survivors in BC.NAD(P)H:quinone oxidoreductase 1(NQO1),also known as DT-diaphorase,is a cytoplasmic flavoenzyme encodedby a gene located on chromosome 16q22.NQO1 uses NADH or NADPH as substrates to directly reduce quinones to hydroquinones.Functions of NQO1 include xenobiotic detoxification,superoxide scavenging and the maintenance of endogenous antioxidant vitamins.It is conceivable that NQO1 plays an important role in protecting normal cells against oxidative injury and carcinogenesis.Paradoxically,despite the cellular functions of this "cell protector",the antioxidant role of NQO1 was suggested by evidence that the disruption of the NQO1 gene or genetic polymorphism increased the risk of chemical-induced toxicity and cancers.In recent years,the role of NQO1 in many kinds of malignant tumors has attracted much attention of scholars.Ma et al.reported that NQO1 expression was higher in SCCs of the cervix than in the normal cervical epithelia,and closely related to the differentiation,clinical stage and lymph node metastasis.Zhang et al.demonstrated that NQO1 overexpression inhibits the proliferation and induces apoptosis of HCC cell by activating the AMPK/PGC-1α pathway.Additionally,Cheng et al.had proven that NQO1 could potentiate NSCLC cell proliferation by enhancing cellular glycometabolism,and NQOl depletion triggered metabolic shift of TCA cycle independent of PDHX and PDK.These studies indicated that NQO1 was an oncogene,and the high-level expression of NQO1 is associated with multiple malignant tumors progression.Our previous studies found that NQO1 can be used as an early diagnostic and prognostic indicator for BC,but its role and mechanism in the progression of BC remains unclear.Therefore,it is urgent to study the mechanism of NQO1 on biological behavior of BC,and provide an effective theoretical basis for targeted therapy of BC.Objectives:This study aimed to identify the casual role of NQO1/PKLR axis as a tumor promoter in regulating the process of BC.(1)To explore the clinicopathological significance of NQO1 overexpression and provide an effective therapeutic target for BC.(2)To investigate the effects of NQO1 regulating on the growth,metastasis and glycolysis reprogramming in BC cells and explore the molecular mechanism of NQO1 in BC progress.(3)To reveal the correlation between NQO1 and metabolic gene PKLR,and further identify the mechanism of NQO1/PKLR axis in regulating the process of BC.Materials and methods:(1)Clinical specimens and database analysis:TCGA cohort and GDS were used to analysis the expression levels of NQO1 mRNA in BC tissues.TCGA cohort and The Human Protein Atlas were used to verify the value of evaluation of NQO1 in prognosis of the BC patients.NQO1 protein expression in 296 cases of BC and 95 normal tissues were detected by immunohistochemical method,and the correlations between abnormal expression of protein and clinicopathological characteristics were also analyzed.(2)Experiments in vitro:The lentivirus-mediated NQO1 upregulating and silencing system were used to modulate NQO1 expression in BC cells.MTT,colony formation and EdU assays were used to detect the effects of NQO1 modulation on cell proliferation.Wound healing and transwells assays were used to determine the effects of NQO1 modulation on cell migration and invasion.Metabolic Kits were used to detect the effects of NQO1 modulation on cell glucose,lactate,and ATP.Western blot and IF were used to detect the expression of epithelial markers and mesenchymal markers in BC cells.Western blot was used to detect the expression of metabolic markers in BC cells.Co-Immunoprecipitation(CoIP)and si-RNA transfection assay were used to detect the underlying mechanisms between NQO1 and PKLR.(3)Experiments in vivo:Different groups of BC cells were injected subcutaneously into the nude mice,and compared the tumor formation and proliferation in different groups.The model of metastatic tumor in vivo was established by injecting the tail vein of nude mice,and compared the lung metastasis in different groups.The expression levels of Ki67,NQOl,PKLR,E-cadherin,Vimentin in tumor tissue of nude mice were detected by IHC method.The lung metastases were analyzed by hematoxylin-eosin(HE)staining.Results:(1)NQO1 overexpression correlates with poor prognosis in BC:NQOl showed a mainly cytoplasmic staining pattern in breast cancer,and the levels of NQOl mRNA were significantly up-regulated in BC tissue samples.The positive rates of NQO1 in breast cancer tissues(85.5%)was significantly higher than in normal breast tissues(27.4%),and were related with clinical stage,pathological grade,LN metastasis and Her2 status;TCGA cohort and The Human Protein Atlas showed that abnormal expression of NQOl was closely related with BC survival(p<0.05).(2)NQO1 enhances tumor growth and in vivo tumorigenesis:MTT and clonogenic assays demonstrated that NQO1 overexpression enhances BC cells growth.EdU assay showed that NQO1 can regulate DNA replication ability of cells.In vivo experiments further confiramed that the high expression of NQO1 could promote the subcutaneous tumor formation of nude mice.(3)NQO1 induces epithelia-mesenchymal transition and promotes cellular migration and invasion:Wound healing and transwells assays showed that NQO1 overexpression promotes the migration and invasiveness of BC cells.Compared with those in the control group,mice injected with shNQO1 had fewer lung metastases and with NQO1 had more lung metastases.Western blots and IF assays showed that the protein levels of epithelial markers were increased and the mesenchymal markers were significantly reduced in NQO1-silenced cells,whereas the epithelial markers were decreased and mesenchymal markers were significantly increased in NQO1 overexpression cells.(4)NQO1 promoted the malignant progression of BC cell via regulating glycolysis reprogramming:Knockdown of NQO1 strongly decreased the glucose utilization,lactate production,ATP levels and the protein levels of metabolic markers in MCF-7 and Hs-578T cells,whereas overexpression of NQO1 expression decreased the increase utilization,increased lactate production,ATP levels and the protein levels of metabolic markers in MDA-MB-231 and MDA-MB-468 cells.Blocking glycolytic pathway using special inhibitors significantly attenuated NQO1-enhanced proliferation,migration and invasion of BC cells.More importantly,2-DG or of 3-BrPA treatment inhibited the activation of Vimentin,snail,slug and Twist as well as activation of E-cadherin by NQO1.(5)NQO1/PKLR axis regulate the proliferation,EMT process,and glycolysis reprogramming:PKLR acts a target gene of NQO1 in BC cells.Silencing of PKLR using a siRNA approach led to clearly repressed cellular proliferation,migration,and invasion in NQO1 overexpression cells.Western blot also shown that silencing of PKLR decreased expression of mesenchymal markers(Vimentin,slug,snail)and metabolic markers(G6PC,PKM2,HK3),and the elevated E-cadherin level.Positive correlation between NQO1 and PKLR was found in BC cells and tissues.These datas suggested that NQO1/PKLR axis is an important regulator of BC development and progression.Conclusions:(1)NQO1 overexpression is associated with histological grade,clinical stage,LN metastasis,Her2 status,and poor prognosis of BC patients.(2)NQO1 induces epithelia-mesenchymal transition and promotes cellular migration and invasion.(3)The high expression of NQO1 is involved in glycolysis reprogramming in BC,and inhibited the EMT process by glycolysis reprogramming.(4)The NQO1/PKLR axis is an important regulator of BC development and progression,and mainly regulates the metastasis of BC by modulating glycolysis reprogramming.