Gper Regulating ASCT2 Via PI3K/Akt Signaling Pathway Promotes Tamoxifen Resistance In Breast Cancer

Objective:To explore the function of G protein-coupled estrogen receptor(GPER)-upregulated alanine-serine-cysteine transporter 2(ASCT2)and glutamine metabolism on tamoxifen(TAM)resistance in estrogen receptor positive(ER~+)breast cancer.Methods:Western Blot and immunofluorescence(IF)were used to detect the expression and localization of GPER in ER~+breast cancer cells MCF-7 and tamoxifen-resistant cells MCF-7R.Treated with TAM,GPER specific inhibitor G15+TAM and GPER specific agonist G1 respectively,differential gene expressions of MCF-7R were analyzed by gene microarrays.The expression of seven key metabolic enzymes was verified by qRT-PCR and Western Blot.Gene set enrichment analysis(GSEA)and KEGG(The Kyoto Enrichment of Genes and Genomes analysis)of GSE17705,which contains 298 breast cancer patients treated with TAM for more than 5 years were performed.Treated with TAM,the mitochondrial membrane potential of MCF-7 and MCF-7R cells was detected by JC-1 kit,and the ratio of cells with high mitochondrial membrane potential was detected by flow cytometry.After TAM,GPER specific agonist G1 and GPER specific inhibitor G15+TAM were used to activate or inhibit GPER,or LY294002 and PD98059 were used to inhibit PI3k/AKT and MAPK/ERK signaling pathways respectively,the expression of ASCT2 was detected by qRT-PCR and Western Blot,and the consumption of glutamine was detected by glutamine kit.Treated with GPNA or glutamine-free medium,the migration,cell cycle,proliferation and apoptosis of MCF-7R cells were detected by transwell,flow cytometry and CCK-8 kit.Results:1.Western Blot and IF confirm the abnormal activation of GPER in ER+breast cancer cells acquired TAM resistance.Gene microarrays and bioinformatics analysis show that GPER regulates the expression of some energy metabolism-related genes,and aberrant expressions of gene set“GO GLUTAMINE FAMILY AMINO ACID METABOLIC PROCESS”are related to tamoxifen resistance.2.JC-1staining shows enhanced mitochondrial membrane potential of MCF-7R cells treated with TAM.3.qRT-PCR and Western Blot verify that TAM and G1 induce GPER activation,activate PI3K/Akt signaling pathway,up-regulate downstream ASCT2 expression and enhance glutamine uptake;inhibiting GPER activation or PI3K/Akt signaling pathway down-regulates ASCT2 expression and weakens glutamine uptake;inhibiting MAPK/ERK signaling pathway,ASCT2 expression does not change significantly;4.Limitation of glutamine uptake resultes in decreased migration and proliferation of MCF-7R cells and increased sensitivity to TAM.Conclusion:In ER~+breast cancer TAM resistant cells,TAM activates GPER and up-regulates ASCT2 via PI3K/Akt signaling pathway,which results in increased glutamine metabolism and enhanced mitochondrial membrane potential,and promotes TAM resistance in breast cancer.The changes of energy metabolism regulated by GPER play an important role in promoting endocrine therapy resistance.