Study On The Role And Mechanism Of ENO1 As A Metabolic Molecular Switch In Regulating The Stem-like Properties In Gastric Cancer Cells

Gastric cancer(GC)is the fifth most prevalent malignant neoplasm and the third most deadly carcinoma worldwide.Although medical treatment is constantly developing and the treatment methods for GC are constantly updated,the five-year survival rate of GC patients is less than 30%because of tumor aggressiveness,metastasis,chemotherapy resistance,and relapse.Recent studies have demonstrated that gastric cancer stem cells(CSCs)are a rare sub-group of GC cells,which are closely related to malignant behaviors such as tumor metastasis,recurrence,and drug resistance.Due to the existence of CSCs,the cancer cell population exhibits stem-like characteristics,prompting a series of malignant events.In the present study,we demonstrated that the glycolytic enzyme Enolase 1(ENO1)was involved in the regulation of the stem cell-like characteristics of GC cells,as compared to the parental cells,the expression of ENO1 in spheroid cells from cell lines PAMC-82 and SNU16 markedly increased.We used retroviral transduction technology to stably knock down and overexpress ENO1 in PAMC-82 and SNU16 cells and then observed that ENO1 could enhance the stem cell-like characteristics of GC cells,including self-renewal capacity,cell invasion and migration,chemoresistance,and even the tumorigenicity of GC cells.We found that ENO1 could increase the expression of the stem cell markers CD44,Nanog,Oct4,and Sox2 in GC cells.Taken together,these results suggested that ENO1 could enhance the CSC-like characteristics of GC cells.Moreover,we used AP-Ⅲ-a4(ENOblock)to inhibit the function of ENO1 in PAMC-82 and SNU16 cells and then investigated the changes of stemness of GCs,we found that ENOblock could inhibit the stemness of GC cells,and the function of inhibition by ENOblock was in consistent with the function of ENO1-knockdown.We further confirmed that ENO1 could promote the stemness of GC cells,ENO1 was the important molecule to maintain gastric cancer stem cells(GCSCs).ENO1 was known as an enzyme that is involved in glycolysis,but we found that ENO1 could independently regulate the level of glycolysis in GC cells and was an important molecular switch in the glycolysis pathway.Our results showed that ENO 1 could significantly increase the glucose consumption,lactic acid production and the extracellular acidification rate(ECAR)of GC cells.Taken together,we confirmed ENO1 could markedly promote the glycolytic activity of GC cells.Subsequently,to further confirm our results,we found that the inhibition of ENO1 using ENOblock could reduce the glycolytic activity of GC cells.Furthermore,inhibiting glycolysis activity using 2-Deoxy-D-glucose(2-DG)treatment significantly reduced the stemness of GC cells,including self-renewal capacity,cell invasion and migration,and chemoresistance.Moreover,2-DG was used to treat GC cells stably overexpressing and knocking down ENO1.We found that 2-DG could restore the stemness changes of GC cells caused by changes in ENO1 expression.Therefore,ENO1 could improve the stemness of GC cells by enhancing the glycolysis.ENO1 increased the production of ATP by promoting glycolysis in GC cells.Since AMPK could be activated by the increasing AMP/ATP ratio,we investigated the changes of AMPK in stable GC cells that overexpress and knock down ENO 1,and the changes in AMPK of GC cells after ENOblock or 2-DG treatment.We found that ENO1 could promote the glycolysis in GC cells and increase ATP production,thereby inhibiting the activation of AMPK,which in turn activated mTOR.We used AMPK’s agonist AICAR to treat GC cells that overexpressing ENO1 and found that AICAR could partially restore the changes in the stemness characteristics of GC cells caused by changes in ENO 1 expression.Therefore,ENO1 promoted the stemness of GC cells in part by regulating AMPK.In addition,we found that the addition of exogenous lactate to parental cells could significantly promote the stemness of GC cells by promoting the PI3K/AKT/mTOR pathway.We tested the changes of PI3K/AKT/mTOR pathway in stable GC cells that overexpress and knock down ENO1,and the changes of PI3K/AKT/mTOR pathway of GC cells after ENOblock or 2-DG treatment.We found that ENO1 could promote the glycolysis in GC cells and increase the production of lactate,thereby activating the PI3K/AKT pathway and then activating mTOR.We used PI3K’ inhibitor LY294002 to treat GC cells that overexpressing ENO1 and found that LY294002 could partially restore the changes in the stemness of GC cells caused by changes in ENO1 expression.We used PI3K1 agonist 740Y-P to treat stable GC cells that knock down ENO1,and found that 740Y-P could partially restore the changes in the stemness of GC cells caused by changes in ENO1 expression.Therefore,ENO1 promoted the stemness of GC cells in part by regulating the PI3K/AKT pathway.Since both AMPK and PI3K/AKT pathways could regulate mTOR,we used mTOR1 inhibitor Rapamycin to treat GC cells that overexpress ENO1 and found that Rapamycin could almost completely restore the changes in the stemness of GC cells caused by changes in ENO1 expression;we used mTOR’ agonist MHY1485 treated GC cells that knocked down ENO1,and found that MHY1485 also could almost completely restore the changes in the stemness of GC cells caused by changes in ENO1 expression.Therefore,ENO1 could increase the production of ATP and lactate by promoting the glycolysis level of GC cells,thereby regulating the synergistic effect of AMPK/mTOR and PI3K/AKT/mTOR pathways to promote the stemness of GC cells.Finally,we analyzed the clinical relevance of ENO1 using tissue chips prepared from clinical specimens and found that the higher the expression of ENO1,the lower the survival rate of patients,and the lower the expression of ENO1,the higher the survival rate of patients.Therefore,increased expression of ENO1 was related with poor prognosis in GC patients.Taken together,our results demonstrated that ENO1 is a significant biomarker associated with the stemness of GC cells and the new target of GC therapy.In conclusion,ENO1 could increase the production of ATP and lactate by promoting the level of glycolysis in GC cells,thereby inhibiting the activation of AMPK and promoting the activation of PI3K/AKT.The two synergistically promoted the activation of mTOR,which in turn promoted the stem-like properties of GC cells.