The Function And Mechanism Of SIX1 In Glycolysis

Cancer cells exhibit aberrant metabolism characterized by high glycolysis even in the presence of abundant oxygen.This phenomenon,known as the Warburg effect or aerobic glycolysis,facilitates tumor growth with elevated glucose uptake and lactate production^[1-7].The Warburg effect has now been widely accepted as a hallmark of cancer and cancer therapeutic agents targeting the Warburg effect are being developed.Over ten genes encoding glycolytic enzymes are directly responsible for the Warburg effect^[8-12].Transcription factors play a direct role in regulation of the Warburg effect^[13-14].Only a few transcriptional factors that have been reported to be associated with glycolysis,such as HIF,c-Myc,NF-kB and p53.Hypoxia-inducible factor-1α（HIF-1α）is a transcriptional activator that acts as a key regulator of the Warburg effect^[15,16].HIF-1αincreases expression of the majority of glycolytic genes by binding hypoxia-responsive elements of glycolytic gene promoters.The c-Myc oncogenic transcription factor directly transactivates glycolytic genes encoding glucose transporter1（GLUT1）and lactate dehydrogenase A（LDHA）,and stimulates aerobic glycolysis^[17,18].In contrast,the p53 tumor suppressive transcription factor directly represses the transcription of GLUT1 and GLUT4 genes,causing decrease in glucose uptake and lactate production^[19,20].Although a few transcription factors have been shown to control the Warburg effect,transcriptional regulation of the Warburg effect remains largely unknown.Transcription factor sine oculis homeobox 1（SIX1）is a key regulator of organogenesis^[21,22].Six1 knockout（KO）mouse embryos have defects in several organs and are relatively smaller in size than wild-type littermates^[23,24].Six1 KO mice die shortly after birth.SIX1 is overexpressed in many cancers^[25-31],such as breast cancer,prostate cancer,hepatocellular carcinoma,and colorectal cancer.Increased expression of SIX1 predicts poor clinical outcomes.SIX1 promotes tumor growth and metastasis through regulation of cell-cycle progression and epithelial-mesenchymal transition^[32-34].However,whether SIX1 regulates cancer metabolism is unclear.In this study,transcriptome RNA sequencing showed that SIX1 regulated glycolytic genes and participated in glycolysis pathway.Quantitative real-time PCR,Western blot,luciferase activity test and Ch IP assay showed that we show that SIX1 is the major transcription factor activating the transcription of the majority of glycolytic genes and promoting aerobic glycolysis.Mechanistically,the interaction between SIX1and histone acetyltransferase HBO1 and AIB1^[35-38]was demonstrated by immunoprecipitation,GST pull-down,or His pull-down.Quantitative real-time PCR and Western blot indicated that SIX1 promoted glycolysis and tumor cell growth through interaction of HBO1 or AIB1.Luciferase activity test showed that miR-548a-3p bound the 3’-UTR of SIX1 directly.Western blot,glycolysis product assay and growth curve test showed that miR-548a-3p inhibited many glycolytic genes expression,glycolysis and tumor cell growth by inhibiting SIX1.Moreover,the miR-548a-3p/Six1axis regulates tumor growth largely through the Warburg effect.Western blot,glycolysis product assay,growth curve test and nude mouse tumorigenicity assay testified that cancer-related SIX1 mutation^[39,40]increased its ability to promote the Warburg effect and tumor growth.¹⁸F-PET imaging showed that the glucose uptake of Six1 KO fetal mice was significantly lower than that of WT fetal mice.Clinically,high expression of SIX1 and low expression of miR-548a-3p in breast cancer patients were associated with high glucose uptake.Immunohistochemical analysis proved that SIX1was positively correlated with expression of the glycolysis genes PGK1 and LDHA,and miR-548a-3p was negatively correlated with SIX1,PGK1 and LDHA.The analysis of clinical data showed that low expression of miR-548a-3p indicated poor prognosis of breast cancer patients.Thus,the miR-548a-3p/SIX1 axis closely links glycolysis to tumor.It provides a potential way for developing anticancer drugs by targeting glucose metabolism and it also provides useful information about the prognosis of malignant tumors.