TOP1MT-deficiency Promotes GC Invasion And Migration Via The Enhancements Of LDHA Expression And Aerobic Glycolysis

Purpose and Background:Metabolic disturbance is a universal property of cancer cells and one of the most important causes of tumorigenesis and cancer progression.The main metabolism-related characteristic of most cancer cells is a metabolic switch from the mitochondrial tricarboxylic acid(TCA)cycle to aerobic glycolysis.Even under aerobic conditions,glucose is taken up and transformed to lactate via the glycolytic pathway in cancer cells,a phenomenon known as the Warburg effect.This metabolic switch produces an acidic microenvironment for the tumor and promotes the epithelial-mesenchymal transition(EMT)and metastasis,leading to poor prognosis.Studies of the Warburg effect in tumors have been a major focus of tumor metabolism research.Therefore,analysis of the Warburg effect and exploring the underlying target genes may provide clues for therapeutic strategies in clinical oncology.Experimental Design:12 fresh gastric cancer(GC)specimens were examined by qRT-PCR and Western-blotting methods.The expression of Toplmt in gastric cancer specimens were analyzed.By the same method the expression of Toplmt were tested in 8 gastric cancer cell lines.In gastric cancer cell lines,to investigate the function of invasion and metastasis in gastric,scratch assay and Transwell migration assay were performed.TOP1MT gene interferencec and animal models were used to clarify the role of TOP1MT in gastric cancer.GC cells were transiently transfected with siRNA targeting TOP1MT.Mechanically,by TOP1MT gene silencing,the glucose consumption and lactate production,the production of ATP and glucose transporter 1 expression of TOP1MT were detected in gastric cancer cell glucose metabolism(including glycolysis and aerobic oxidation).Finally,In order to further demonstrate the role of TOP1MT in GC,we analyzed the clinical data of 295 GC patients with follow-up data on clinical,immunohistochemical method was used to analyze the expression and clinical significance of TOP1MT in gastric cancer tissues.Results:.We first detected the RNA expression of TOP1 MT in tissue by RT-PCR and found that TOP1MT mRNA levels were lower in GC tissues than in adjacent normal tissues.Western blotting results also showed that TOP1MT expression was significantly lower in GC tissue samples.We further examined TOPI MT expression in seven human GC cell lines and found that TOP1MT expression was lower at both mRNA and protein levels in GC cell lines compared with that in GES-1 cells.To explore the effects of low TOP1MT expression on GC cells,we performed functional experiments in BGC-823 and SGC-7901 cells.Cells were transiently transfected with siRNA targeting TOP1MT.To demonstrate that the shTOPIMT constructs specifically targeted the mitochondrial isoform of TOPI,mRNA and protein levels of TOPI,TOP2a,TOP2P,TOP3a,and TOP3β were tested by RT-PCR and western blotting.TOP1MT knockdown did not affect the mRNA or protein levels of these targets in GC cells.In transwell assays,the numbers of migrating and invading cells were significantly higher for siRNA-transfected BGC-823 and SGC-7901 cells.In addition,wound-closure migration assays also showed that TOP1MT silencing significantly accelerated wound recovery.To determine the effects of low TOP1MT expression in vivo,TOP1MT-silenced(shTOP1MT)BGC-823 and SGC-7901 cells and their corresponding negative controls,BGC-823/scramble and SGC-7901/scramble,were established.TOP1MT knockdown increased pulmonary metastasis in both cell lines,as demonstrated by anatomical pathology and histopathology.These results clearly indicated that TOP1MT silencing promoted the invasion and metastasis of GC cells.TOP1MT silencing significantly increased glucose utilization,lactate production,and ATP generation and decreased the pH of the supernatants in BGC-823 and SGC-7901 cells.Increased glucose transporter 1(Glut1)expression further verified the upregulation of glucose metabolism in cells with TOP1MT silencing.These results indicated that glycolysis was enhanced in GC cells with TOP1MT silencing.To further explore the effects of TOP1MT knockdown on mitochondria in GC cells,we detected cytochrome C oxidase(COX)expression as a marker of OXPHOS activity in mitochondrial respiration.Compared with the control,siTOP1MT significantly decreased the mRNA and protein expression of COX in GC cells.LDHA mRNA and protein expression and LDHA activation were significantly increased in BGC-823 and SGC-7901 cells with TOP1MT knockdown.Immunohistochemical staining results of subcutaneous xenograft tumors showed that TOP1MT knockdown enhanced LDHA expression in vivo,consistent with the results of the GC cell lines in vitro.Our results showed that sodium oxamate or siLDHA counteracted the increased migration and invasion induced by TOP1MT silencing.The results of wound-closure migration assays further verified these results.The inhibition of LDHA by sodium oxamate or siLDHA counteracted the increased wound recovery induced by TOPIMT silencing.We also found that sodium oxamate or siLDHA reversed the enhancement of glucose utilization and lactate production and the decrease in pH values of the supernatants in BGC-823 and SGC-7901 cells induced by TOP1MT silencing.RT-PCR and western blotting results showed that in both shTOP1MT/BGC-823 and SGC-7901 GC cell lines,the expression levels of vimentin and fibronectin were significantly higher,whereas E-cadherin expression was significantly lower than those in negative control cells.Next,we examined whether LDHA was associated with changes in EMT markers induced by TOP1MT silencing in vitro.We found that TOP1MT silencing upregulated vimentin and fibronectin mRNA and protein and downregulated E-cadherin mRNA and protein.However,inhibition of LDHA by sodium oxamate counteracted these effects in both BGC-823 and SGC-7901 cells.TOP1MT expression was significantly decreased in GC specimens relative to normal gastric samples by immunohistochemical staining using monoclonal anti-TOP1MT antibodies.Moreover,TOP1MT expression was significantly lower in patients with GC showing lower differentiation,more regional lymph node metastasis,more advanced stage disease,and recurrence in stages Ⅰ-Ⅲ,and in cases of death in patients with stage Ⅳ disease during follow-up.Immunohistochemical staining of LDHA in consecutive sections for these 295 patients with GC showed that LDHA expression was significantly higher in patients with GC having lower differentiation or more regional lymph node metastasis and in cases of death in patients with stage Ⅳ disease during follow-up.The disease-free survival rates of patients with stage Ⅰ-Ⅲ cancer and the overall survival rates of patients with stage Ⅳ cancer were shorter in patients with low TOP1MT expression than in patients with high TOP1MT expression.By analyzing consecutive GC sections,we found that TOP1MT expression was significantly associated with LDHA expression.These clinical results were consistent with the results of our in vitro and in vivo experiments using GC cell lines.Conclusion:In this study,in vitro and in vivo experiments showed that low expression of TOP1MT promoted the invasion and metastasis of GC cells;the possible mechanism for TOP1MT deficiency in GC cells increased glucose aerobic glycolysis,especially the expression of LDHA,inhibited the aerobic oxidation of glucose.This study also showed that EMT may be an important factor in the invasion and metastasis of GC cells with TOP1MT deficiency.In conclusion,our study revealed the TOP1MT played important role in the invasion and metastasis in GC cells,and inhibition metabolism by interfering with TOP1MT express may provide clues for therapeutic strategies in clinical oncology.