the tumorigenesis of cancer cells involves numerous genetic and epigenetic lesions , resulting in the critical alteration of multiple cellular restraints . many oncogenes may induce persistent activation of unusual cell procedures and promote carcinoma growth [ 1 , 2 ] . it is widely accepted that tumor cells shift their metabolism away from respiration toward anaerobic glycolysis , to achieve excessive cell growth , proliferation , and resistance to apoptosis . most tumor cells exhibit increased glycolysis and take this metabolic pathway as a main source of their energy supply to generate atp . thus , the increasing of glycolysis of tumor cells may be recognized as an important event in tumorigenesis and potential target for antitumorigenesis for cancer therapy . nowadays , tremendous progress has been made in understanding of the molecular mechanisms of tumorigenesis , especially in the signaling corresponded for its increased glycolysis , such as pi3k - akt - mtor signaling [ 5 , 6 ] . however , it is still largely unknown for the complicated network of increasing glycolysis of tumorigenesis . astrocyte elevated gene-1 ( aeg-1 ) is originally cloned as a gene induced in primary human fetal astrocytes ( phfa ) infected with hiv-1 or treated with tumor necrosis factor- ( tnf- ) . it is also revealed that aeg-1 was significantly elevated in subsets of breast carcinoma , melanoma , and malignant glioma cell lines compared to their normal cell counterparts . the correlation of aeg-1 with many carcinomas affords the potential that aeg-1 plays a crucial role in the oncogenic transformation and tumorigenesis [ 7 , 8 ] . previous study showed that aeg-1 expression was markedly induced by ha - ras , which was transcriptionally mediated through the pi3k signaling pathway [ 9 , 10 ] . other reports revealed aeg-1 functions in cell transformation , which is an important event in tumorigenesis [ 1113 ] . it was also demonstrated that aeg-1-expressing tumors have increased microvessel density throughout the entire tumor sections , which also contributes to the tumorigenesis . in vitro angiogenesis studies further reveal that aeg-1 promotes tube formation in matrigel and increases invasion of human umbilical vein endothelial cells with increased expression of angiogenesis markers , such as hypoxia - inducible factor 1- ( hif-1 ) [ 7 , 9 , 15 , 16 ] . it is noted that aeg-1 may increase the expression of hif-1 , which is a critical factor for glycolysis [ 17 , 18 ] . besides hif-1 , aeg-1 is also proved to activate ampk , which may also be of potential to promote glycolysis and tumorigenesis [ 8 , 13 , 19 ] . amp - activated protein kinase ( ampk ) is a highly conserved heterotrimeric kinase complex and activated under conditions of energy stress . the phosphorylation of the activation loop threonine is absolutely required for ampk activation [ 20 , 21 ] . it is found that aeg-1 reduces the atp / amp ratio and activates amp kinase , which induces ampk - dependent autophagy [ 13 , 22 , 23 ] . interestingly , aeg-1-induced autophagy by ampk protects cells from apoptosis , which might benefit tumor cells for surviving under pharmacological conditions . although aeg-1 exhibits several regulatory functions in tumorigenesis , it is intriguing whether there are other aspects of tumorigenesis regulated by aeg-1 and how aeg-1 activates intracellular signaling pathways . in the present study , we showed that aeg-1 may upregulate glycolysis in ncm460 human colonic epithelial cells by activation of ampk signaling . the activated ampk then phosphorylates 6-phosphofructo-2-kinase ( pfk-2 ) and induces the synthesis of fructose 2,6-bisphosphate , which is a key intermediate product of glycolysis . moreover , inhibition of ampk activity in ncm460 cells or interfering aeg-1 expression in hct116 human colon carcinoma cells reversed the increased glycolysis . all these results will help to understand a novel clue regarding the role of aeg-1 as a potential cancer therapeutic in tumor cells , as well as the molecular mechanism of tumorigenesis . dulbecco 's modified eagle 's medium ( dmem ) , trypsin , and fetal bovine serum ( fbs ) were obtained from hyclone ( hyclone , logan , utah ) and m3 media from sigma ( st . louis , mo , usa ) . the anti - aeg-1 antibody was from epitomics ( san diego , ca , usa ) . anti - beta - actin was purchased from millipore ( billerica , ma , usa ) . pampk ( t172 ) and amp - activated protein kinase ( ampk ) antibody were from cell signaling technology ( beverly , ma , usa ) , while ppfk2 ( s466 ) and phosphofructokinase-2 ( pfk2 ) were from santa cruz ( santa cruz , california , usa ) . the ampk inhibitor compound c was provided by merck ( billerica , ma , usa ) . ncm460 cell line was a nontransfected human colonic epithelial cell line , while hct116 cell line was established from a primary colon carcinoma and widely used in the colon cancer research . the ncm460 cells were introduced from incell corporation ( san antonio , tx , usa ) and hct116 , ht29 , and sk / s cells from the american type culture collection ( manassas , va , usa ) . ncm460 cells were cultured in m3 media and hct116 in dmem with 10% fetal bovine serum ( fbs ) and 1% antibiotics at 37c in a 95% air/5% co2 atmosphere at constant humidity . following , transfection was carried out when the cell confluent was 8090% using lipofectamine 2000 , and cell was harvested at 48  h after transfection with lysis buffer ( pbs+1% triton + proteinase inhibitors ) . all the transfection manipulations , including aeg-1 overexpression and sirna , were performed as transient transfection , not stable clones . for compound c treatment , 20  m compound c was applied to cells and lasted for 2  h to block ampk activity . the myc - aeg-1 construct for overexpression of aeg-1 in ncm460 cells was generated by subcloning the pcr - amplified human aeg-1 coding sequence into prk5-myc vectors . to reduce the endogenous aeg-1 protein level in hct116 cells , we generated another aeg-1 rnai psuper vector with the following oligonucleotides , 1 # aacagaagaagaagaaccgga and 2 # gaaatcaa agtcagatgcta ( invitrogen ) as previous report . western blot was performed as standard procedures . to extract proteins , cultured cells were sonicated with lysis buffer ( pbs ) with 1% triton x-100 and proteinase inhibitors . the protein concentration of each extract was measured by the bca protein assay kit ( thermo scientific pierce , rockford , il , usa ) . equal amounts of the proteins from each extract were loaded into and separated by sds - page . the membranes were then blocked by 5% nonfat dry milk in tbst ( tbs with 0.1% tween 20 , ph 7.6 ) for 1  h at room temperature and probed overnight by proper primary antibodies diluted in tbst at 4c . after 3 times of washing with tris - buffered saline and tween ( tbst ) at room temperature for 10  min each , the membranes were incubated with proper secondary antibodies diluted in tbst ( 1  :  10,000 for both goat anti - rabbit and goat anti - mouse igg ) for 1  h at room temperature . after another 3 times of membrane washing with tbst at rt for 10 minutes each , proteins were detected by pierce ecl western blotting substrate kit ( thermo scientific / pierce , rockford , il , usa ) reagent and exposed to film ( kodak ) . total rnas were extracted from cells using trizol reagent ( invitrogen , carlsbad , ca , usa ) . rna was subjected to reverse transcription with reverse transcriptase as manufacturer 's instructions ( fermentas inc . quantitative real - time pcr was performed using the bio - rad iq5 system using bio - rad proprietary iq5 software ( hercules , ca , usa ) , and the relative gene expression was normalized to internal control as beta - actin . primer sequences for sybr green probes of target genes were as follows : aeg-1 : ttgaagtggctgagggtgaa and tacgctgctgtcgtttctct and beta - actin : gagaccttcaacaccccagc and atgtcacgcacgatttccc . a total of 68  10 cells per well were seeded in 12-well plates for 24  h and then treated with or without compound c for 48  h. cell numbers were counted before measurements using a hemocytometer . glucose was measured spectrophotometrically using an olympus au5400 ( olympus corporation , tokyo , japan ) . the lactate was measured using a rxl max system ( dade behring , deerfield , il , usa ) . all results of western blots , real - time pcr , and biochemical assays across time were presented as mean  sem . from three independent experiments at least . p values were calculated using student 's t - test for normally distributed data , and the values 0.05 (  ) , 0.01 (  ) , and 0.001 (  ) were assumed as the levels of significance for the statistic tests carried out . numerous reports have revealed the essential role of aeg-1 in the development and progression of cancer . aberrant elevation of aeg-1 expression frequently occurs in human cancers , including breast cancer , glioma , melanoma , esophageal squamous cell carcinoma , prostate cancer , hepatocellular carcinoma , and gastric cancer . moreover , aeg-1 is found to be differentially overexpressed in adenoma and cancer cells , whereas it is weakly expressed in normal mucosa [ 8 , 25 , 26 ] . although aeg-1 is potentially correlated with colorectal carcinomas , it is not well understood how aeg-1 performs in colorectal carcinomas . thus , the mrna and protein levels of aeg-1 in colorectal carcinomas were investigated in canonical human colon carcinoma cell lines , such as hct116 , ht29 , and s / ks . the protein levels of aeg-1 in hct116 , ht29 , and s / ks cells were significantly increased than that of ncm460 cells , by 16.2- , 13.4- , and 15.9-fold ( figures 1(a ) and 1(b ) ) . results of real - time pcr analysis also showed that the transcriptional level of aeg-1 was extremely high in these three colorectal carcinoma cells , increased by 18.7- , 20.1- , and 22.3-fold in hct116 , ht29 , and s / ks cells compared to that of ncm460 ( figure 1(c ) ) . the extremely high transcription of aeg-1 affords its protein translation in colorectal carcinoma cells , which contributes to the tumorigenesis transcriptional homeostasis impairment from many aspects . to study the role of aeg-1 in colon cancers , we next generate and validate the myc - tagged aeg-1 overexpression vectors and after transfection , the ncm460 cells were harvested for subsequent western blot , to confirm the overexpression of myc - aeg-1 . the myc blots indicated that human - derived aeg-1 was expressed in ncm460 cells ( figure 2(a ) ) . performing metabolic assays of glucose consumption and lactate production in aeg-1-overexpressed ncm460 cells , the present results indicate that glucose consumption and lactate production in aeg-1-overexpressed cells increased by 4.6- and 4.8-fold compared with those in nontransfected control cells , respectively , and the values show significant differences ( figures 2(b ) and 2(c ) ) . this consistent increasing of glucose consumption and lactate production is a typical feature of anaerobic glycolysis and suggests that aeg-1 may promote anaerobic glycolysis . it has been reported that aeg-1 may cause a significant increase of ampk phosphorylation at thr172 . while the phospho - ampk ( thr172 ) may activate ampk activity and phosphorylate 6-phosphofructo-2-kinase at ser466 sites , which may induce the synthesis of fructose 2,6-bisphosphate , a potent stimulator of glycolysis . therefore , we assume that aeg-1 upregulates anaerobic glycolysis by increasing ampk / pfk2 axis . to test this , we find that ampk phosphorylation at thr172 is increased in ncm460 cells overexpressed of aeg-1 ( figure 2(d ) ) . however , the total level of ampk showed no significant change . for ampk may promote glycolysis by phosphorylating pfk2 , we next check the pfk2 phosphorylation at ser466 in ncm460 cells with aeg-1 overexpression . similarly , the ppfk2 ( ser466 ) also increases in aeg-1-overexpressed cells ( figure 2(d ) ) . we have described that aeg-1 may increase the phosphorylation of ampk and pfk2 and promote anaerobic glycolysis . to confirm these findings , we design to inhibit ampk activity and test whether this will reduce pfk2 phosphorylation and anaerobic glycolysis under aeg-1-overexpressed conditions . the activity of ampk could be inhibited by compound c. it is shown that compound c may antagonize aicar ( ampk activator ) by blocking the uptake of aicar into cells . although aeg-1 activates ampk phosphorylation , compound c could block ampk phosphorylation in both control and aeg-1-overexpressed ncm460 cells . pfk2 also showed similar results and aeg-1 may not activate pfk2 phosphorylation in the presence of compound c ( figure 3(a ) ) . as expectedly , compound c drastically reduced aeg-1-induced glucose consumption and lactate production by ~38.7% in aeg-1 nontransfected cells . moreover , compound c could further decrease the glucose consumption and lactate production by ~48.2% and ~49.4% compared to aeg-1-transfected cells ( figure 3(b ) ) . thus , these findings confirmed that aeg-1 may promote anaerobic glycolysis by ampk - pfk2 axis , and inhibition of ampk may reverse aeg-1-mediated increasing glycolysis . it was shown that aeg-1 was highly expressed in human colon carcinoma cells and enhanced anaerobic glycolysis via ampk in ncm460 cells . we proposed that ectopic expression of aeg-1 in colon carcinoma cells was responsible for the activated anaerobic glycolysis . to test this hypothesis , we constructed aeg-1 rnai psuper vectors to decrease the endogenous aeg-1 expression in hct116a widely used human colon carcinoma cell line . then , we examined the aeg-1 protein levels and found that the aeg-1 expression was decreased in these transfected cells than that of controls ( figure 4(a ) ) . next , we examined the glucose consumption and lactate production and found that they were inhibited by aeg-1 rnai ( figure 4(b ) ) . moreover , our results suggested that decreased aeg-1 protein levels may inhibit cell migrations in hct116 cells . this result confirmed that blocking energy production by aeg-1 rnai indeed impaired tumor metastasis ( figure 4(c ) ) . based on the above results , these findings support the notion that aeg-1 may be critical for anaerobic glycolysis in colon carcinoma cells , and the inhibition of aeg-1 expression may restore the ectopic anaerobic glycolysis in these tumor cells . astrocyte elevated gene-1 ( aeg-1 ) expression increases in multiple cancers and plays a crucial role in oncogenic transformation and angiogenesis , which are essential components in tumor cell development , growth , and progression to metastasis . moreover , aeg-1 directly contributes to resistance to chemotherapeutic drugs , another important hallmark of aggressive cancers [ 8 , 12 ] . multitissue northern blots containing total rna of normal human organs revealed that aeg-1 mrna is ubiquitously expressed at varying levels in all organs . expression analysis in cell lines revealed that aeg-1 expression is significantly higher in breast , prostate , esophageal , and liver cancer and melanoma , malignant glioma , and neuroblastoma cell lines in comparison to their normal counterparts [ 11 , 14 , 2831 ] . these studies provide important insights and a unique perspective on this multifunctional oncogene , aeg-1 . in the present study , we demonstrated a novel mechanism that aeg-1 might increase the anaerobic glycolysis through the activation of ampk signaling . and inhibition of ampk activity or downregulation of aeg-1 expression may reverse the effects of aeg-1 on glycolysis ( figure 4(d ) ) . anaerobic glycolysis is a metabolic process in which glucose , a sugar molecule , is broken down without the use of oxygen . the phenomenon of anaerobic glycolysis increasing in cancer cells is first described by otto warburg ( 1930 ) over 70 years ago . many cells ranging from microbes to lymphocytes use aerobic glycolysis during rapid proliferation , suggesting that it may play a fundamental role in supporting cell growth . results showed that malignant cells exhibit significantly elevated glycolytic activity even in the presence of sufficient oxygen and consider this phenomenon as the most fundamental metabolic alteration in malignant transformation , or  the origin of cancer cells  [ 4 , 32 ] . although the cause - effect relationship between the increase in anaerobic glycolysis and the development of cancer is controversial , increased glycolysis has been consistently observed in many cancer cells of various tissue origins . indeed , the positron emission tomography ( pet ) widely used in clinical diagnosis of cancer is based on the fact that cancer cells are highly glycolytic and actively uptake glucose [ 33 , 34 ] . the warburg effect can be viewed as a prominent biochemical symptom of cancer cells that reflects a fundamental change in their energy metabolic activity . several mechanisms have been suggested to affect energy metabolism and thus contribute to the warburg effect . these mechanisms include ( 1 ) mitochondrial defects , ( 2 ) adaptation to hypoxic environment in cancer tissues , ( 3 ) oncogenic signals , and ( 4 ) abnormal expression of certain metabolic enzymes . it seems that malignant cells become additive to glycolysis and dependent on this pathway to generate atp . because atp generation via glycolysis is far less efficient ( two atp per glucose ) than through oxidative phosphorylation ( 36 atp per glucose ) , cancer cells consume far more glucose than normal cells to maintain sufficient atp supply for their active metabolism and proliferation [ 35 , 36 ] . this metabolic feature has led to the hypothesis that inhibition of glycolysis may severely abolish atp generation in cancer cells and thus may preferentially kill the malignant cells . recent studies have provided supporting evidence that inhibition of glycolysis may exert preferential effect on cells with compromised mitochondrial function due either to genetic defects or a lack of oxygen [ 37 , 38 ] . during periods of intracellular metabolic stress , a reduced cellular energy ( atp ) level previous reports showed that aeg-1 induces noncanonical autophagy involving an increase in expression of atg5 . aeg-1 decreases the atp / amp ratio , resulting in diminished cellular metabolism and activation of amp kinase , which induces ampk / mammalian target of rapamycin - dependent autophagy . in the present study our work shows that inhibition of aeg-1-ampk axis by pharmacological treatment ( such as compound c ) may be of potential for the clinical biotherapy of tumorigenesis . in summary , we identified aeg-1/ampk / pfk2 signaling for anaerobic glycolysis in human colon carcinoma cells . it is speculated that the frequent hyperactivation of aeg-1/ampk signaling in the course of the multistep oncogenesis may contribute to the development of the warburg effect present in many human cancers . our work may provide a mechanistic insight into novel targets controlled by aeg-1 , and the components in aeg-1/ampk / pfk2 signaling in glycolysis may be targeted for the treatment of cancer .
to investigate the role of aeg-1 in glycolysis and tumorigenesis , we construct myc - aeg-1 expression vector and demonstrate a novel mechanism that aeg-1 may increase the activity of ampk by thr172 phosphorylation . 
 the higher expression levels of aeg-1 in colorectal carcinoma cells were found but showed significant difference in different cell lines . to study the role of aeg-1 in colorectal cells , 
 myc - aeg-1 vector was constructed and transfected into ncm460 colonic epithelial cells . 
 we observed consistent increasing of glucose consumption and lactate production , typical features of anaerobic glycolysis , suggesting that aeg-1 may promote anaerobic glycolysis . 
 moreover , we noted that ampk phosphorylation at thr172 as well as ppfk2 ( ser466 ) was increased in ncm460 cells overexpressing aeg-1 . 
 compound c may block ampk and pfk2 phosphorylation in both control and aeg-1-overexpressed cells and decrease the glucose consumption and lactate production . 
 the present findings indicated that reduced aeg-1 protein levels by rnai may decrease the glucose consumption and lactate production in hct116 colorectal carcinoma cells . 
 the present identified aeg-1/ampk 
 / pfk2 glycolysis cascade may be essential to cell proliferation and tumor growth . 
 the present results may provide us with a mechanistic insight into novel targets controlled by aeg-1 , and the components in the aeg-1/ampk / pfk2 glycolysis process may be targeted for the clinical treatment of cancer .