Effects Of GPER-mediated Glutamate Secretion On Invasion And Metastasis Of Triple Negative Breast Cancer(TNBC)and Its Mechanism

Objective: To investigate the effect of GPER-mediated estrogen on invasion and metastasis of TNBC cells and its related mechanisms.Methods:(1)Using mRNA and lncRNA microarrays to detect and analyze potential GPER-regulated mRNAs and lncRNAs.And qRT-PCR was used to verify the results of mRNA and lncRNA microarrays.The analysis of signal pathway enrichment was carried out with DAVID v.6.8 KEGG database.(2)The expression of VGLUT2 in TNBC samples and adjacent normal tissues was detected by qRT-PCR.Immunohistochemistry(IHC)was used to detect the expression of GPER and VGLUT2 in TNBC tissues.The correlation between GPER and VGLUT2 was analyzed by scoring IHC results.Western blotting was used to verify the relationship between GPER and VGLUT2 in TNBC cells.Glutamate levels released by TNBC cells during GPER activation and inhibition were detected using glutamate assay kit.(3)The level of lncRNA-Glu in 96 TNBC patients was detected by qRT-PCR to verify the relationship between GPER and lncRNA-Glu in TNBC cells.The binding of lncRNA-Glu to VGLUT2 was verified by RNA pull-down assay and RNA immunoprecipitation assay(RIP).The effect of lncRNA-Glu binding to VGLUT2 on glutamate transport activity was analyzed by glutamate uptake assay.The effect of lncRNA-Glu on the transcriptional activity of VGLUT2 was analyzed by luciferase reporting assay.(4)The relationship between GPER and cAMP-PKA signaling in TNBC cells was investigated using cAMP detection kit and western blotting.Glutamate assay kit was used to verify the relationship between GPER-cAMP-PKA signaling and glutamate levels,the relationship between GPER-lncRNA-Glu-VGLUT2 signaling and glutamate levels,and the relationship between the two signaling pathways.Through cell invasion assay,the relationship between GPER-cAMP-PKA signaling,GPER-lncRNA-Glu-VGLUT2 signaling and TNBC cell invasion ability was verified.(5)IHC was used to detect the levels of NMDAR subunit NR2 b in TNBC and the correlation between NR2 b and GPER.Western blotting was used to verify that increased glutamate secretion promoted NMDAR receptor activation and CaMK/ MEK-MAPK signaling activation.The effect of CaMK/MEK-MAPK signaling activation on cell invasion ability was verified by cell invasion assay.The level of MMP7 was detected by qRT-PCR.(6)Orthotopic xenograft mouse models were employed to verify the results of our in vitro experiments.H&E staining was used to obtain the lung metastasis burden of mice under different treatment conditions.The levels of lncRNA-Glu and VGLUT2 were detected by qRT-PCR.Glutamate concentration in xenograft tumors of mice was determined by glutamate assay kit.Western blotting was used to verify the status of NMDAR-cAMK/MEK-MAPK signals in xenograft tumors of mice.Results:(1)We obtained 589 potential GPER-regulated mRNAs.And 2102 potential GPER-regulated lncRNAs and 625 predicted target genes.These 589 mRNAs and 625 target genes were enriched in several signaling pathways known to be associated with cancer cell invasion respectively,among which we found the glutamate signaling pathway and the vesicle glutamate transporter 2(VGLUT2)in this pathway.The mRNA microarray showed that VGLUT2 was upregulated by E2 and G1 and downregulated by G36 and GPER depletion.(2)VGLUT2 was highly expressed in TNBC and positively correlated with GPER.Consistent with the mRNA microarray results,in vitro experiments showed VGLUT2 was upregulated by E2 and G1,and downregulated by G36 and GPER depletion.At the same time,glutamate concentration was increased by E2 and G1,and decreased by G36 and GPER depletion.(3)VGLUT2 is the target gene of a novel lncRNA(termed as lncRNA-Glu in this paper).In TNBC cells,lncRNA-Glu level was decreased by E2 and G1,and increased by G36 and GPER depletion.LncRNA-Glu was downregulated due to the activation of GPER,and the downregulated lncRNA-Glu will increase glutamate transport activity and transcriptional activity of VGLUT2.(4)GPER-mediated activation of cAMP-PKA signaling was observed in TNBC cells.In addition,GPER-cAMP-PKA signaling and GPER-lncRNA-Glu-VGLUT2 signaling can synergistically promote glutamate secretion and TNBC cell invasion.(5)The increased glutamate secretion can promote the phosphorylation of NMDAR subunit NR2 b,thereby activating the NMDAR receptor and CaMK/MEK-MAPK signaling,causing the phosphorylation of CREB,thereby recruiting CBP,increasing the expression of its target gene MMP7,and promoting the invasion of TNBC cells.(6)There were notable surface lung metastases in mice treated with E2 or G1;whereas MDL and MK801 treatment attenuated the G1 stimulated tumor metastases;lncRNA-Glu overexpression reduced G1-mediated tumor metastases(knockdown of endogenous lncRNA-Glu could partly rescue the metastases),and inhibition of cAMP signaling using MDL further decreased surface lung metastases in mice;however,exogenous supplementation of glutamate to mice could partly rescue the surface lung metastases.In vivo experiments demonstrated that the GPER-glutamate signaling pathway promotes TNBC cell invasion and metastasis.Conclusion: LncRNA-Glu was downregulated by GPER activation.LncRNA can inhibit glutamate transport activity and the transcriptional activity of VGLUT2,so the downregulated lncRNA activated by GPER can enhance the glutamate transport activity and the transcriptional activity of VGLUT2.At the same time,lncRNA-Glu-VGLUT2 signaling and GPER-cAMP-PKA signaling synergistically increase the glutamate secreted by TNBC cells.Increased glutamate can activate its receptor NMDAR and cause activation of downstream CaMK/MEK-MAPK signaling,thus enhancing the invasion and metastasis ability of TNBC cells.The mechanism of GPER-mediated TNBC invasion and metastasis we found provides new potential targets for the clinical treatment of TNBC.