The Mechanism Of JAC4 Regulating Energy Metabolism And Metastasis Of Pancreatic Cancer Cells

Pancreatic cancer(PC)is known as the "king of cancer" and its mortality rate is ranked 7th worldwide.The incidence of pancreatic cancer in China is ranked 10 th and the mortality rate is ranked 6th in 2015.Chemotherapy is the standard therapy for advanced or metastatic PC,but the efficacy is very limited and the mortality rate remains high.The development of cancer is related to the signal network disorder and caused by either inactivation of tumor suppressor molecules or excessive activation of cancer-promoting molecules or by both in cancer cells.FOXO3a is a member of the FOXO forkhead transcription factor subfamily that mediates a variety of cellular processes including apoptosis,proliferation,cell cycle,DNA damage and repair,and tumorigenesis;it also responds to several cellular stresses such as UV radiation and oxidative stress.The function of FOXO3 a is regulated by complex networks,including post-transcriptional inhibition of micro RNAs(mi RNAs),post-translational modifications(PTM),and protein-protein interactions.FOXO3 a is distributed widely in vary organs and cells and has a wide range of biological effects,thus involving the development of various diseases including cancer.FOXO3 a is a recognized tumor suppressor,and is usually inactivated by gene mutation or cytoplasmic chelation of protein;and its inactivation is associated with the occurrence and development of cancer.The role of oncogenes or tumor suppressor genes in the development of cancer usually involves interactions or regulatory networks with other genes and proteins;therefore,elucidating the molecular basis of these interactions and regulating the nature of the network is an urgent need for development of cancer diagnosis and therapy.The JWA gene,also known as ARL6ip5,is an active environmental response gene whose protein is localized in endoplasmic reticulum and mitochondria,and is a cytoskeletal binding protein.JWA is a known multifunctional protein,involves in DNA damage and repair,apoptosis,cell migration,angiogenesis and metastasis;in addition,JWA has been shown to function as a tumor suppressor gene,plays an essential role in promoting cancer cell apoptosis and inhibiting cancer cell adhesion,angiogenesis and metastasis.However,whether JWA gene involves in the development and progress of pancreatic cancer is not clear.In this study,gene transfection and small molecule agonist JAC4 were used to increase the expression level of JWA in cells,and to investigate its role and molecular mechanism in regulating the migration and metastasis of pancreatic cancer cells.Objective: To elucidate the role and molecular mechanism of JWA in regulating cell energy metabolism through FOXO3 a and inhibiting the migration and metastasis of pancreatic cancer cells.Methods: Western blotting was used to detect the expressions of associated milecules including JWA,FOXO3 a,AMPK and FAK in pancreatic cancer cells.The pancreatic cancer cells Panc1 and Bxpc3 were used to eslatblish series in vitro cell culture models and in vivo metastatic mice models.Transfection of either Flag-JWA plasmid or si RNA of JWA was used to increase or reduce the expressions of JWA gene;the JWA agonist JAC4 was used to treat cells or mice to increase JWA expression.The assays of wound healing and transwell were used to determine cell migration;the mitochrondria energy metabolisms of cells were measured by Seahorse XF energy metabolism analyzer.The role of JAC4 in inhibiting the in vivo growth and metastasis of pancreatic cancer was further determined by PDX mice modes and passive metastasis,respectively.Result:1.The expression of JWA gene is reduced in pancreatic cancer tissues.Using TCGA data analysis,we found that the expression level of JWA was significantly lower in pancreatic cancer tissues than that in normal pancreatic tissues(P =0.022).2.JWA inhibits the migration of pancreatic cancer cells.The wound healing assay showed that the cell migration rate was reduced by 52% after high expression of JWA in Panc1 cells compared with the control group;and the cell migration rate was increased by 117% after the low expression of JWA in Bxpc3 cells.Transwell assay showed that the number of perforated cells was reduced by 35% after high expression of JWA in Panc1 cells compared with the control group;and the number of perforated cells was increased by 44% after low expression of JWA in Bxpc3 cells.3.JAC4 inhibits the migration of pancreatic cancer cells.Panc1 and Bxpc3 pancreatic cancer cells were treated with 0,0.1,1,10,50 μM JAC4 for 48 h.Western blotting confirmed that JAC4 treatment increased the expression level of JWA in both cells,and with a dose-dependent relationship.The IC50 value of JAC4 on Panc1 and Bxpc3 pancreatic cancer cells was 204 and 655 μM,respectively.The results of Transwell assay showed that the migration ability of both pancreatic cancer cells was inhibited by JAC4 at 10 μM for 48 h compared with the control group;as a fact,the migrations in Panc1 cells were reduced by 46% and in Bxpc3 cells by 32%,respectively.The wound healing assay showed that after treatment of JAC4 at 10 μM for 48 h,the migration capacity of Panc1 and Bxpc3 cells was 66% and 35% of the solvent control group,respectively.4.JAC4 prolongs the survival of pancreatic cancer PDX mice and reduces serum LDH levels.The four human pancreatic cancer tissues were used to construct mice PDX models;the 18 model mice were divided into 3 groups(6 mice/case/treatment;72 mice were used for 4 cases)and treated by solvent control,chemotherapeutic drug(gemcitabine + paclitaxel)positive control and JAC4,respectively for 21 days.The results showed that the survival of mice in 3 groups was 21/24,19/24 and 24/24,respectively.In addition,blood biochemistry assay showed an obvious change of LDH levels amomg groups.As a fact,the serum LDH levels of mice were significantly decreased in both chemotherapeutic drug positive control(579.2±112.86 U/L)and JAC4(630.75±137.89 U/L)groups compared to the mice in solvent control group(885.78±231.63 U/L).5.The effect of JWA on energy metabolism in pancreatic cancer cells.The expression level of JWA protein in Panc1 cells was increased by transfection or JAC4 treatment for 48 h;and the relationship between JWA expression level and cell energy metabolism was determined by Seahorse XF extracellular energy metabolism analyzer.The results showed that JWA increased aerobic respiration(basal respiration 1.55 times and 3.22 times,ATP production 1.35 times and 1.36 times,maximal aerobic respiration 2.05 times and 2.64 times),and however inhibited glycolysis(21%)and glycolytic capacity(8%)of Panc1 cells(gene transfection had no significant effect on tumor glycolysis),and with dosedependent manners in both aspects.6.JWA positively regulates FOXO3 a but negatively regulates FAK expressions in Panc1 cells.Panc1 cells were treated by transfection or JAC4 for 48 h.Western blotting showed that the level of JWA was significantly increased,and the expressions of both FOXO3 a and p-FOXO3 a were increased correspondingly;however the expression level of FAK decreased(45% and 49%,respectively);after knocking down of JWA in Bxpc3 cells,the expressions of both FOXO3 a and pFOXO3 a were also decreased,while the expression level of FAK was increased by 1.32 times.7.JAC4 via FOXO3 a inhibits migration of pancreatic cancer cells by stimulating mitochondrial complex III.Western blotting showed that the expression level of mitochondrial complex III(UQCRC2)was significantly increased(1.42 fold by transfection,1.54 fold by JAC4 treatment,respectively),and there was no significant effects on the expression levels of other 4 mitochondria complexes(I,II,IV,and V).Panc1 cells were treated with either JAC4 or JAC4 combined with FOXO3 a si RNA for 48 h,and then treated with complex III inhibitor anti-mycin A(500 n M)for 1 h.Western blot results showed that FAK expression was 3.04 times higher in JAC4 combined with FOXO3 a si RNA group than in JAC4 treatment alone.However,after co-treatment with JAC4 and FOXO3 a si RNA alone or JAC4 10 μM alone,treatment with antimycin A increased the expression of FAK by only 1.98 times.It is suggested that the inhibitory effect of JWA on FAK is achieved through the FOXO3a-mitochondrial complex III pathway.8.JWA positively regulates FOXO3 a expression via the AMPK signaling pathway.Panc1 cells were treated with JAC4 for 48 h and then treated with 10 μM compound C for further 6 h.Western blotting showed that the expression level of JWA was increased,and the expression trends of FOXO3 a and p-AMPK were consistent with the trend of JWA;however,the trend between FAK,m TORC and JWA was opposite,and there was no significant change in AMPK.After JAC4 and compound C treatment,the expression levels of AMPK and p-ampk were decreased,FOXO3 a expression was inhibited,but the expression level of FAK was increased.Panc1 cell migration was increased by the use of compound C,as demonstrated by perforation.It suggested that compound C effectively inhibited the activation of AMPK and downstream FOXO3 a,and further blocked the inhibition of JAC4 on cell migration through FAK.9.JAC4 inhibits pancreatic cancer metastasis in mice and reduces serum LDH levels.Panc1 cells(5×105/100 μL PBS)were inoculated into the tail vein of nude mice to establish a passive metastasis model,and divided as control group and JAC4 intervention group.The results showed that the number of metastatic mice in the control group and JAC4 treatment group was 5/14 and 3/15,respectively.Compared with the control group,the lung metastasis area/right lung area(under HE staining)was 2.5 times low in the JAC4 treatment group,and the lactate dehydrogenase level was lower in the JAC4 group than that in the control group(P <0.01).Conclusion:JWA positively regulated FOXO3 a through AMPK signaling pathway,activated oxidative phosphorylation of pancreatic cancer cells,and inhibited its glycolysis;JWA played an active role in inhibiting the migration and metastasis of pancreatic cancer cells.The results provided new academic basis for elucidation of the molecular mechanism of JWA inhibiting the migration and metastasis of pancreatic cancer cells.It also provided new idea for the development of new therapies targeting tumor suppressor gene JWA in the treatment of pancreatic cancer metastasis.