Yin Yang 1 Promotes The Warburg Effect And Tumorigenesis Via Glucose Transporter GLUT3

Tumor metabolism is one of the hot topics in cancer research in recent years.In the condition of adequate oxygen supply,normal cells uptake glucose as the primary source of cellular energy,and metabolite it through oxidative phosphorylation.Specifically,after entering the cell,glucose will be convert to pyruvate,and through the tricarboxylic acid cycle and oxidative phosphorylation,pyruvate will be metabolized subsequently to carbon diaoxide and water in the mitochondria.This reaction generates more than 30 ATPs.However,tumor cells prefer to use glycolytic enzymes to metabolize glucose and produce energy,altough the ATPs generated from glycolytic pathway is much lesser than oxidative phosphorylation.This is due to the rapid growth of tumor cells,which requires a large number of various biosyntheic intermediates produce by glycolytic pathways to provide the cells with the building blocks needed for generation of cellular compartments.Thus,tumor cells prefer glycolytic pathway rather than oxidative phosphorylation.Glucose is the main energy source for cell,and its an indispensible material for cell metabolism.Glucose is transported into the tumor cells by the glucose transporter.Currently,there are 14 members,that is,GLUT1-14,of the GLUT family that have been identified.These glucose transporters are distributed in different tissues,with distinct substrate selectivity.Among them,GLUT3 is a glucose transporter which is normally expressed in skeletal muscle cells and other specific tissues;however,its expression is upregulated in various tumor tissues.GLUT3 is also a glucose transporter with very high affinity for glucose(Km = 1.5 mM),which is approximately 5 times of the Km of GLUT1,which is ubiquitously expressed.Yin Yang 1 is an oncogene that could function as a transcriptinal factor.YY1 could promote or inhibit the transcriptional activity of its target protein,depends on the context it binds.Previous studies,including our own,have shown that YY1 is closely related to tumor angiogenesis,tumor cells proliferation and tumor cells DNA repair,and thus induces tumorigenesis.YY1 could regulate the expression levels of GLUT1,p53,HIF-1?,c-myc and other factors critical for tumorigenesis.YY1 is overexpressed in various kinds of tumor,including breast cancer,ovarium cancer,cerebral cancer,colon carcinoma and melanoma.In this study,we constructed shRNA expression vector against YY1,and knocked-down YY1 expression in wild-type and p53 null colon cancer cells HCT116.knockdown plasmid was constructed to knock down YY1 in p53-wild and null-type colon cancer cell HCT116 cells.We found that YY1 silencing significantly suppressed GLUT3 mRNA and protein expression levels.YY1 silencing also suppressed the glucose uptake and lactate production of the HCT116 cells,as well as cell proliferation.These effects of YY1 silencing could be rescued by the overexpression of GLUT3,which restored glucose uptake,lactate production and cell proliferation potential suppressed by YY1 silencing.Besides,by using dual luciferase reporter assay,we found that YY1 could enhance the promoter activity of GLUT3,and thus promotes GLUT3 expression level.Furthermore,by performing chromatin immunoprecipitation assay and luciferse assay using GLUT3 promoter with mutations on the predicted YY1 binding site,we found that YY1 could binds to GLUT3 promoter,and thus induce GLUT3 transcriptional activity.Thus,our study revealed,for the first time,that YY1 could positively regulate the expression of glucose transporter GLUT3,enhance tumor cells' glucose uptake and lactate production,and subsequently,promotes tumor cells proliferation and tumorigenesis.Furthermore,this regulation of YY1 on GLUT3 expression level occurs through a direct binding of YY1 to GLUT3 promoter,which then enhances GLUT3 transcriptional activity.Finally,by performing xenograft experiment using p53 null colon cancer cells in which YY1 is knocked-down,we demonstrated in vivo that YY1 positively regulate tumorigenesis potential by regulating GLUT3 expression.We found that tumorigenesis potential of this p53 null colon cancer cells was significantly reduced by YY1 silencing;however,overexpression of GLUT3 could restored it.At the same time,we also examined the expression levels of YY1 and GLUT3 in clinical sampels of human colon carcinoma tissues,and found that the mRNA and protein expression levels of both YY1 and GLUT3 were significantly higher than in normal tissues.Together,these results indicate that YY1 promotes glycolysis and tumorigenesis by regulating GLUT3 in a p53-independent manner.Additionally,we also found that oxaliplatin could suppress colon carcinoma cells proliferation through inhibiting YY1/GLUT3 pathway.In summary,our findings revealed that YY1 is a critical regulator of tumor cells glucose metabolism that regulates the expression of GLUT3,the crucial factor of glucose uptake,which is the pacesetter of the glycolysis.These results not only unravel a novel role of YY1 in regulating tumor cells metabolism,but also provides molecular basis for the potential of using YY1 as a molecular target for tumor therapeutic strategy.