Molecular Mechanism By Which The Glycolysis Inhibitor 2-DG Inhibits ERK Phosphorylation

Lung cancer is one of malignant tumors with highest morbidity and mortality in our country.Non-small-cell lung cancer(NSCLC)accounts for about 80%?85%of all types of lung cancer.At present,the 5-year survival rate of NSCLC is only about 15%.Better understanding of the molecular mechanism underlying lung cancer development is of great significance for the diagnosis and treatment of lung cancer.Cells need energy to survive and maintain normal function.The main material to provide energy is glucose.Glucose metabolism,including glycolysis and oxidative phosphorylation,produces a large amount of ATP and plays an important role in cellular metabolism.One of the primary hallmarks of cancer is that tumor cells are more dependent on the glycolytic metabolism to generate ATP,namely "Warburg"effect.Over the years,targeting glycolysis has been explored as a therapeutic approach for cancer treatment.Of all the glycolysis inhibitors that were evaluated,2-deoxyglucose(2-DG)is the one that has been best characterized in animal model studies and human clinical trials.It is converted by hexokinase to 2-DG-P,which becomes trapped inside the cell and inhibits hexokinase,the rate-limiting enzyme in glycolysis.As a direct consequence of 2-DG treatment,intracellular ATP is depleted.The inhibition of glycolysis by 2-DG treatment exerts indirect effects on various signaling pathways.For example,2-DG treatment inhibits mammalian target of rapamycin(mTOR)signaling,which is mediated by LKB1/AMP-activated protein kinase(AMPK)signaling,an energetic stress-sensing signaling pathway.Specifically,the inhibition of glycolysis by 2-DG treatment leads to a decrease in intracellular ATP concentration and an increase in intracellular AMP concentration.AMP binds to AMPK and alters AMPK conformation,which results in AMPK activation by the upstream regulator LKB1.Activated AMPK then phosphorylates TSC2 and raptor,thus inhibits mTOR activity.In addition,2-DG has also showed“off-target" effects,which are independent of the inhibition of glycolysis or LKB1/AMPK signaling.Our group previously discovered that 2-DG treatment disrupted the binding between insulin-like growth factor 1(IGF-1)and IGF-binding protein 3(IGFBP3),and increasd the free form of IGF-1 to activate IGF-1 receptor(IGF 1R)signaling,thus resulting in enhanced downstream RAF-MEK-ERK and PI3K-AKT signaling.In an effort to investigate the effect of 2-DG on the signaling pathways downstream of IGF1R,we observed an intriguing phenomenon.We found that 2-DG induced ERK phosphorylation is only observed in LKB1 mutant NSCLC cells.In LKB1 wildtype cells,surprisingly,2-DG treatment led to a decrease in the level of P-ERK,in a time and dose-dependent manner.Whereas 2-DG induced AKT activation is observed in both LKB1 mutant cells and LKB1 wildype NSCLC cells.These results strongly suggest that the presence of LKB1 might function in inhibiting ERK phosphorylaiton.LKB1,a serine/threonine kinase,is frequently mutated in lung cancer.As a tumor suppressor,LKB1 plays significant roles in inhibiting lung cancer progression and metastasis.Next,we investigated the potential involvement of LKB1 in ERK regulation.We found that ectopic expression of LKB1 in LKB1 mutant NSCLC cells significantly attenuated 2-DG induced ERK activation.In addition,we established LKB1 stable knockdown isogenic NSCLC cells using Lenti-virus system,and found that LKB1 knockdown led to increased level of P-ERK.These results showed that LKB1 negatively regulates ERK phosphorylation.As a key substrate of LKB1,AMPK plays a key role in the regulation of cellular metabolism,cell growth and cell polarity.Next,we examined the role of AMPK in ERK inhibition.We found that the suppressing AMPK activity by Compound C greatly blocked 2-DG mediated ERK inhibition.In addition,inhibiting AMPK expression by siRNA resulted in enhanced P-ERK level.Moreover,overexpression of AMPK with constitutive activity(CA-AMPK)inhibited ERK phosphorylation.Taken together,the data indicated that AMPK functions downstream of LKB1 to negatively regulate ERK activation.Therefore,we proposed that 2-DG inhibits ERK phosphorylation by activating LKB1/AMPK signaling.RAF/MEK/ERK signaling is activated when cytokines like EGF or IGF1 binds with the receptors.Next,we investigated the effect of 2-DG on cytokine induced ERK activation.We found that 2-DG was able to inhibit ERK activation both in the absence and presence of fetal bovine serum(FBS).Specifically,2-DG significantly decreased IGF1 induced ERK phosphorylaiton,while it had little effect on EGF induced ERK activation.These results suggest that 2-DG induced LKB1/AMPK activation might negatively regulate the IGF1R signaling and thus inhibits ERK phosphorylation.Ras is the main upstream regulator of RAF/MEK/ERK signaling.As a key oncogene,it is frequently mutated and activated in lung cancer.We subsequently sought to investigate the effect of Kras mutation activation on 2-DG mediated ERK inhibition.We found that 2-DG mediated ERK inhibition was observed only in Kras wildtype isogenic colon cell lines,but not in Kras G13D cells.In addition,overexpression of Kras G12V plasmid in Kras wildtype cells also blocked 2-DG mediated ERK inhibition.These data indicated that wildtype Kras is required for ERK inhibition by 2-DG,and 2-DG activated LKB1/AMPK might function at the Ras level or upstream Ras to inhibit ERK activation.In summary,our study showed that the glycolysis inhibitor 2-DG activates LKB1/AMPK,which negatively regulates IGF1R signaling and Ras activity,and inhibits ERK phosphorylation.These findings help to better understanding the mechanism of action of 2-DG and provide rational for lung cancer personalized therapy.Moreover,the study also reveals the potential cross-talk between LKB1/AMPK and ERK signaling,and offer novel insights into the tumor suppressor role of LKB1.