SIPA1/HIF-2α Promotes Carcino-metastasis Via Activating Aerobic Glycolysis In Breast Cancer

Cancer cells rely heavily on high level of glycolytic fermentation for energy metabolism even with ambient oxygen supply,which is called aerobic glycolysis or the Warburg effect.This abnormal metabolic pathway provides rapid energy production and abundant metabolic intermediates for tumor survival and growth,and contributes to tumor infiltration and metastasis by reprogramming the microenvironment.Previous studies indicate that signal-induced proliferation associated 1(SIPA1)alteres tumor microenvironment and promotes breast cancer metastasis.This study aims to investigate the mechanism how SIPA1 regulates aerobic glycolysis to drive breast cancer cell metastasis.⑴ SIPA1 enhanced aerobic glycolysis and inhibited mitochondrial activity in breast cancer cells.Analysis of the GSE9014 and GSE41313 datasets showed that the expression of aerobic glycolytic genes such as HK2,PKM2,and LDHA was higher in triple-negative breast cancer(TNBC)cells than that in the Luminal subtype.Expression of SIPA1 showed a significant positive correlation with multiple glycolytic genes such as ENO1,LDHA and PKM2.In addition,SIPA1-high expressed TNBC cells MDA-MB-231,BT549 and SUM159 revealed a significantly higher level of aerobic glycolysis than MCF7,an ER-positive cell line with low SIPA1-expression.These phenomena suggest that SIPA1 may be related to the regulation of aerobic glycolysis in breast cancer cells.To determine this hypothesis,the SIPA1 stably knockdown MDA-MB-231 cell line(231si)and BT549 cell line(549/sh SIPA1),and the MCF7/SIPA1 cell line overexpressing SIPA1 were constructed.Knocking down SIPA1 in the above two TNBC cells decreased the glycolytic flux such as glucose consumption and lactate production,and down-regulated the expression of glycolysis-related genes at m RNA and protein levels,as well as the intracellular and extracellular lactate dehydrogenase activity.While overexpression of SIPA1 in 231 si and MCF7 cells obtained opposite results.In addition,SIPA1 knockdown caused an increase in ATP levels in MDA-MB-231 cells,while SIPA1 overexpression caused a decrease in ATP levels in 231 si and MCF7 cells.Oligomycin,a mitochondrial ATP synthase inhibitor,and sodium pyruvate treatment experiments showed that SIPA1 knockdown reprogrammed the cellular ATP source to shift from glycolysis to mitochondria,and SIPA1 overexpression shifted the cellular ATP source from mitochondria to aerobic glycolysis,which made SIPA1-high expressed MDA-MB-231 and MCF7/SIPA1 sensitive to be inhibited on proliferation by oxamate,an LDH inhibitor,rather than oligomycin.q RT-PCR and western blotting detection demonstrated that PDK1,inactivating the transformation of pyruvate to acetyl-Co A,was positively regulated by SIPA1 in MDA-MB-231 and MCF7 cells.These results indicate that SIPA1 enhanced the Warburg effect by up-regulating expression of glycolysis related genes and inhibited mitochondrial activity by up-regulating PDK1.⑵ Aerobic glycolysis mediated by SIPA1/HIF-2α promoted the migration of breast cancer cells in vitro.After confirming that the protein levels of key glycolysis-regulators HIF-1α and c-myc are not affected by knockdown or overexpression of SIPA1,this study performed transcriptome sequencing on the two pairs of cell lines MDA-MB-231 and231 si,MCF7 and MCF7/SIPA1.The results of differential gene enrichment and GO analysis showed that the genes positively regulated by SIPA1 were significantly enriched in a biological process termed “response to hypoxia” linked by transcription factor HIF-2α(encoded by EPAS1 gene)and its downstream target genes such as CA9 and VEGFA.This group of genes were confirmed positively regulated by SIPA1 in breast cancer cells using q RT-PCR and western blotting.It was further demonstrated by Ch IP-PCR and dual luciferase reporter gene experiments that SIPA1 bound to the promoter region of EPAS1 gene to enhance its transcriptional activity.In addition,knocking down EPAS1 gene reduced the glucose consumption and lactate production of MDA-MB-231 and BT549 cells,and down-regulated the expression of aerobic glycolytic genes such as SLC2A1,HK2,LDHA and PDK1.Scratch and transwell tests revealed that SIPA1 and EPAS1 knockdown and LDH inhibitor oxamate treatment inhibited the migration and infiltration of MDA-MB-231 and BT549 cells in vitro.⑶ SIPA1/HIF-2α promoted the growth and metastasis of breast cancer in vivo,leading to poor prognosis.Animal model analysis revealed that both knockdown of SIPA1 and oxamate treatment strongly suppressed the growth of orthotopic xenografts,concentration of lactate in the orthotopic xenografts and metastatic nudles on the lungs in xenografted nude mice.Consistently,the expression of EPAS1,SLC2A1 and PDK1 in the MDA-MB-231 xenografted tumor were significantly higher than that in the 231 si xenografted tumor.TCGA and GSE41313 dataset analysis showed that SIPA1 was aberrantly expressed in breast cancer tissues and cells,especially in TNBCs.SIPA1 and EPAS1 gene expression are positively correlated in both datasets.Furthermore,the high expression of EPAS1 was associated with the shortened survival of Grade 3 breast cancer patients.These results indicate that activation of SIPA1/HIF-2α pathway may be a factor leading to poor prognosis of breast cancer patients.In summary,this study revealed that SIPA1 enhances EPAS1 expression by binding to its promoter,and up-regulated the expression of genes such as HK2,LDHA and PDK1,which tended to transform the cells into a glycolytic phenotype,thereby driving breast cancer process.Significantly,targeting the SIPA1/HIF-2α axis-mediated aerobic glycolysis pathway may be an effective treatment strategy for patients with invasive breast cancer with high SIPA1-expression.