NFAT5-SLC5A3 Axis Facilitates Pancreatic Ductal Adenocarcinoma’s Warburg Effect Through Inositol-transport

OBJECTIVES: Several datasets were selected to screen for the most glycolysis-related genes in pancreatic ductal adenocarcinoma(PDAC).Determine the expression pattern,clinical significance and biological function of the targent gene in PDAC.Explore the impact on glucose metabolism in PDAC and underlying mechanism.Evaluate the clinical validity of corresponding anatgonist.METHODS: TCGA and GEO datasets were selected for top 10% glycolysis-related gene screening.The expression pattern of target gene SLC5A3 were then analized in gene expression profile array,tissue microarray and KPC mouse tissue,together with the correlation with clinical parameters and follow-up data were used to evaluate the clinical significance.Pancreatic cancer cell lines were utilized for in vitro function assay.Subcutaneous and orthotopic xenograft model were established for in vivo assay.To assess the effect on glucose metabolism,Seahorse XF96 Flux Analyser were used to measure ECAR and OCR.Potential transcription factor were predicted by online database and previous studies.Down stream signal pathway were predicted by Gene set enrichment analysis(GSEA)and validated by Western Blot.The result was further verified by IHC staining for clinical specimen.ELISA assay was used to detect the difference in phosphatidylinositol’s concentration between different cell groups.Orthotopic xenograft model and KPC mouse were established to evaluated the clinical potential of SLC5A3 antagonist phlorizinRESULT: Based on the TCGA and our expression profile array,SLC5A3 was selected as our target gene.The correlation with clinical parameters and follow-up data showed that the expression of SLC5A3 is higher in tumor tissue and act as an independent prognostic predictors.In vivo assay demonstrated that high expression of SLC5A3 could promote the proliferation and prevent the apoptosis of pancreatic cancer cells.In addition,in vitro assay showed the same result.By preforming seahorse glycolysis stress test and cell mito stress assay we found that interfereing SLC5A3 expression in cell lead to decreased ECAR and OCR,indication that SLC5A3 could facilitates the glucose metabolism in PDAC.GSEA showed that hypoxia was enriched in high SLC5A3 expression group.By performing knockdown assay we determined the hypoxia-induced NFAT5 is the potential transcription factor of SLC5A3.GSEA also indicates that PI3K/Akt pathway was enriched in high SLC5A3 expression group.Western and IHC was performed to determine the effect of SLC5A3 expression on Akt phosphorylation and downstream glycolysis-related genes including GLUT1,HK2 and LDHA.CA-Akt plasmid transfecting rescuing the decreasing expression of GLUT1,HK2 and LDHA caused by SLC5A3 interference.ELISA was preformed to detect the intracellular concentration of PIPs and proved that high expression of SLC5A3 increase the concentration of PIPS in cell,which is the main reason for Akt activation.We confirmed by diminishing extracellular uptake of inositol.Then the impact of knockdown of SLC5A3 on PDAC cells was compromised,indicating SLC5A3 promoting tumor progression via transporting inositol.In vivo orthotopic xenograft model and KPC mouse was sacrificed to validated the clinical potential of SLC5A3 antagonis phlorizin.CONCLUSION: Our work demonstrated that inositol transportor SLC5A3over-expressed in PDAC and acts as an independent prognositic factors.Hypoxia-induced transcription factor NFAT5 conduct the transcription of SLC5A3 which then uptake inositol from extracellular environment.Increased inositol level lead to higher amount of PIP3 and facilitates the phosphorylation of Akt.The clinical potential of phlorizin is determined by in vivo orthotopic xenograft model and KPC mouse model.