Effect Of Kras^G12D-LOH On Malignant Phenotypes Of Pancreatic Cancer By Targeting Aerobic Glycolysis And Its Molecular Mechanism

BackgroundKras,one of the major driver genes of pancreatic cancer,has a oncogenic mutation rate of 90%with different mutation types.Our previous research found KrasG12D acquired loss of heterozygosity(LOH),which has only one mutated Kras allele with a loss of the wild type Kras allele,in pancreatic ductal adenocarcinoma mice model.Further study showed KrasG12D-LOH promoted the malignant phenotypes and aerobic glycolysis of pancreatic cancer cells.Moreover,AMP-activated protein kinase(AMPK)and Regulated in development and DNA damage response 1(REDD1)also exhibited higher expression,which revealed AMPK and REDD 1 may be the downstream effectors of KrasG12D-LOH.However,the function of AMPK and REDD1 in aerobic glycolysis and malignant phenotypes driven by KrasG12D-LOH is still unclear.Hence,we aimed to identify the role of AMPK and REDD1 in aerobic glycolysis and malignant behaviors driven by KrasG12D-LOH in vitro and in vivo,and attempted to elucidate the underlying mechanism of this.The will improve the therapeutic efficiency and prognosis of pancreatic cancer.Part OneThe role of AMPK in aerobic glycolysis and malignant phenotypes driven by KrasG12D-LOH in pancreatic cancer cellsAimsTo elucidate the role of AMPK in aerobic glycolysis and malignant behaviors in KrasG12D-LOH pancreatic cancer cells under normoxia and hypoxia.Methods1.Clinical information of pancreatic cancer patients,mRNA levels of AMPK and glycolytic markers in pancreatic cancer samples were collected from The Cancer Genome Atlas database(TCGA).The survival curve analysis were assessed by Log-rank statistical test and Kaplan-Meier analysis.The correlation among AMPK and glycolytic markers were performed by Spearman correlation analysis2.KrasG12D pancreatic cancer cells(hereafter denoted as 399 and 403)and KrasG12D-LOH pancreatic cancer cells(hereafter denoted as 907 and 897)were treated with different doses of AMPK inhibitor Compound C(0μM,0.1μM,1μM,5μM,10μM,20μM),and the most effective dose was selected for further study3.Different groups of cells were incubated at 37℃ under normoxia(95%air,5%CO2)or hypoxia(1%O2,94%N2 and 5%CO2).The cell proliferation was studied by CCK-8 assay and colony formation assay.The cell cycle and apoptosiss were assessed by flow cytometry(FCM).Transwell assay was used to detect the cell migration and invasion4.ELISA kits were used to analyze to the content of aerobic glycolytic products and glucose consumption.Western Blot and realtime-qPCR were used to detect the protein and mRNA levels of aerobic glycolytic markersResults1.Based on TCGA database,185 patients were available for this study.The survival curve showed that patients with high expression of AMPK exhibited a lower median survival time than patients with low expression of AMPK(P<0.05)2.Cells treated with Compound C(10μM)for 24 hours showed significantly reduced phosphorylated AMPK(p-AMPK)protein expression than control group(P<0.05)3.Under normoxia and hypoxia,compared with control group cells,cells treated with Compound C had significantly lower cell proliferation and colony formation ability(P<0.05),their migrated and invaded cell numbers were also decreased(P<0.05).FCM analysis showed that cells treated with Compound C had lower S-phase distribution but higher apoptosis rate than control group cells(P<0.01,P<0.05)4.TCGA data showed all six AMPK subunits(α1,α2,β1,α2,γ1 and γ2)are closely related to different glycolytic biomarkers(P<0.05).And the content of aerobic glycolytic products and glucose consumption in cells treated with Compound C were significantly reduced than those in control group cells(P<0.01,P<0.05,and P<0.05).Moreover,the protein levels of phosphorylated mammalian target of rapamycin(p-mTOR),Pyruvate kinase M2(PKM2)and Hexokinase 2(HK2)were dramatically decreased in Compound C treated cells(P<0.05).Similarly,the mRNA levels were also lower in Compound C treated cells(P<0.05).ConclusionsAMPK can act as an independent factor for prognosis of pancreatic cancer patients,and higher AMPK expression predicts a lower median survival time of patients.Under normoxia and hypoxia,inhibited AMPK activity in two kinds of pancreatic cancer cells can effectively impair the malignant phenotypes,cause changes of cell cycle,promote cell apoptosis,and suppress aerobic glycolysis activity.Those results suggest that AMPK plays an important role in maintaining and supporting the malignant phenotypes in two kinds of pancreatic cancer cells partly by regulating aerobic glycolysis.Part TwoThe role of REDD1 in aerobic glycolysis and malignant phenotypes driven by KrasG12D-LOH in pancreatic cancer cellsAimsTo investigate the role of REDD1 in aerobic glycolysis and malignant events in KrasG12D-LOH pancreatic cancer cells under normoxia and hypoxia.Methods1.Based on the TCGA database,mRNA levels of REDD1 and glycolytic markers in pancreatic cancer samples were collected.The correlation among REDD1 and glycolytic markers were performed by Spearman correlation analysis.2.The REDD 1-shRNA knockdown lentivirus and control lentivirus were transfected into 399 and 897 cells,knockdown efficiency was detected by Western Blot and realtime-qPCR.3.Different groups were incubated under normoxia and hypoxia,assays were performed to analyze the malignant phenotypes.(Methods are the same as part one.)4.399 and 897 cells were treated with different doses(0μM,1μM,2μM,5μM,10μM,20μM)of 2-Deoxy-D-glucose(2-DG)to compare the glycolysis sensitivity.The contents of lactate,pyruvate,ATP and glucose consumption,the protein and mRNA levels of glycolytic markers were detected.(Methods are the same as part one.)5.Pancreatic cancer orthotopic transplantation models in C57BL/6 mice were established by different groups cells.Tumor incidence,metastasis rate,and tumor weight were observed.Immunohistochemistry staining(IHC staining)was used to detected the expressions of Ki67 and PKM2.Results1.Compared with the control lentivirus group(shCon),the protein and mRNA levels of REDD1 in the REDD1-shRNA knockdown lentivirus group(shREDD1)of 399 and 897 cells were significantly lower(P<0.01).2.Under normoxia and hypoxia,compared with shCon group,shREDDl group of 399 cells showed stronger cell proliferation and colony formation ability(P<0.01),and shREDDl group of 897 cells showed lower cell proliferation and colony formation ability(P<0.01).Wound-healing assay and Transwell assay had similar results.FCM data showed shREDDl group of 399 cells had higher distribution of S-phase but lower apoptosis rate(P<0.01),while shREDDl group of 897 cells showed lower S-phase distribution and higher apoptosis rate(P<0.01)3.TCGA data showed REDD 1 had strong correlations with different glycolytic markers(P<0.05).2-DG significantly inhibited cell vitality in 399 cells at 5μM(P<0.05),while in 897 cells at 1μM(P<0.01).And the content of aerobic glycolytic products,glucose consumption,the protein and mRNA levels of glycolytic markers in shREDDl group of 399 cells were significantly increased than control group(P<0.01,P<0.01,P<0.05,and P<0.01),while shREDDl group of 897 cells had opposite results4.In vivo assay showed that compared to control group,shREDDl group of 399 cells exhibited higher metastasis rate,increased tumor weight and higher Ki67 and PKM2 expression(P<0.01,P<0.05,and P<0.01).While shREDDl group of 897 cells showed lower metastasis rate,decreased tumor weight and lower Ki67 and PKM2 expression(P<0.01,P<0.01,and P<0.01)ConclusionsREDD 1 has strong correlation with glycolytic biomarkers,and KrasG12D-LOH renders pancreatic cancer stronger glycolysis dependence.Knockdown REDD 1 expression in KrasG12D pancreatic cancer can improve the malignant behaviors and increase the glycolytic activity.Different from that in KrasG12D pancreatic cancer,knockdown REDD 1 expression can clearly impair the malignant phenotypes of KrasG12D-LOH pancreatic cancer and decrease the glycolysis activity.Those suggest that REDD 1 may function as an important effector in regulating the malignant behaviors of both KrasG12D and KrasG12D-LOH pancreatic cancers.REDD1 suppression can decrease the control of KrasG12D in pancreatic cancer,while REDD1 has sustainable and stimulative function in KrasG12D-LOH pancreatic cancer,and these processes are mediated partly by regulating aerobic glycolysis.