GPER In CAFs Mediates The Energy Metabolism Reprogramming To Regulate Immune Escape Of Triple Negative Breast Cancer In Tumor Microenvironment

Triple-negative breast cancer(TNBC)is known to have a poor prognosis compared to other types of breast cancer.There is no effective therapeutic target for TNBC.GPER(G protein-coupled estrogen receptor)has been shown to play a role in estrogen-mediated effects in TNBC cells,and its activation can promote tumor progression and drug resistance.However,the role of GPER in tumor-associated fibroblasts(CAFs)is unclear.This study will explore the molecular mechanisms that how GPER affects the progression and immune escape by regulating the tumor microenvironment(TME)in TNBC.Part I:The expression and clinical significance of GPER in the tumor microenvironment of advanced TNBCObjective: To investigate the expression of GPER in the breast cancer microenvironment(TME)and its correlation with PD-L1 expression in tumor and clinicopathological variablesMethods: We collected specimens from 91 patients with advanced TNBC who received immunotherapy with PD-1/PD-L1 inhibitors.We analyzed the expression of GPER in the tumor mesenchyme and parenchyme,as well as PD-L1 expression in the tumor parenchyme,and investigated their correlation.We combined GPER expression with clinical information of patients and used Kaplan-Meier method to investigate the association between tumor parenchymal GPER expression,tumor mesenchymal GPER expression,and progression-free survival time(PFS)of patients who received PD-1/PD-L1 inhibitors.Univariate and multivariate Cox analysis were used to identify factors that influenced PFS in patients who received PD-1/PD-L1 inhibitors.Results:(1)High GPER expression in the tumor mesenchyme showed a significant correlation with tumor size(T),regional lymph node metastasis(N),and pathological stage(p<0.05),but no statistically significant correlation with patient age,menstrual status,tumor location,and histological grade(p>0.05);(2)No statistically significant correlation was observed between high expression of GPER in the parenchymal of tumor and age,tumor size(T),regional lymph node metastasis(N),pathological stage,menstrual status,histological grade,and location of the tumor(p>0.05);(3)Spearman correlation analysis showed a positive correlation between GPER expression in tumor mesenchymal and PD-L1 expression in tumor parenchymal(p <0.001,R=0.724).Although a positive correlation was also observed between GPER expression in tumor parenchymal and PD-L1 expression in tumor parenchymal,it did not reach statistical significance(p=0.074,R=0.188);(4)A statistically significant difference(p <0.001)in PFS was observed between the two groups categorized by GPER IHC scores in tumor mesenchymal,with a median PFS difference of 120 days(145 days vs.265 days);(5)The expression of GPER in tumor mesenchymal expression was an independent factor that influenced PFS in patients with advanced TNBC treated with PD-1/PD-L1 inhibitors.Conclusion: The GPER expression in tumor mesenchymal is an independent risk factor for PFS in patients with advanced TNBC treated with PD-1/PD-L1 inhibitors.Part II:Cytoplasmic GPER-mediated cross-talk between cancer-associated fibroblasts and cancer cells governs breast cancer progressionObjective: To explore the molecular mechanism of GPER in tumor-associated fibroblasts(CAFs)regulation of proliferation and invasion in triple-negative breast cancer(TNBC).Methods: Co-culture system of breast cancer cells and CAFs,the function of GPER in CAFs was stimulated by E2(an agonist of GPER)or G15(an antagonist of GPER).Western blotting was used to detect the changes of signal pathway axis related proteins,and CCK8,flow cytometry and invasion test were used to detect the proliferation,apoptosis and invasion of TNBC cells.Glutamine,lactate,ATP,Acetyle-Co A,succinate and glucose production were detected using relevant reagent kits.The binding of GLUL and transcription factors was detected by chromatin immunoprecipitation(Ch IP).Orthotopic xenografts and lung metastasis analysis investigated the biological effects of GPER in TNBC.Results:(1)As cytoplasmic GPER could be activated in CAFs cultured with breast cancer cells and treated with E2 simultaneously,we measured the production of glutamine in intracellular and conditional medium(CM)with or without cytoplasmic GPER activated.The data showed that both intracellular and extracellular glutamine production were enhanced when cytoplasmic GPER activated,and inhibited when G15 existent;(2)By co-culturing BT549 or MDA-MB-231 with CAFs,cell-cycle analysis,cell apoptosis,cell proliferation,cell invasion,and drug resistance were determined.Cell-cycle analysis showed an increase in the fraction of S phase(presented the rate of proliferation)in BT-549 and MDA-MB-231 when treated CAFs with E2.While,the ratio of the fraction of S phase was rescued when knockdown GLUL or treated with G15 in E2-stimulated CAFs.Similarly,breast cancer cell apoptosis decreased,and cell proliferation,cell invasion and drug resistance for epirubicin(EPI)were enhanced as cytoplasmic GPER activated in CAFs.While,there was a converse phenomenon about cell cycle,apoptosis,proliferation,invasion,and drug resistance for EPI as cytoplasmic GPER inactivated or GLUL knockdown in E2-stimulated CAFs;(3)There were an increasing of c AMP production,phosphorylated PKA(p-PKA)expression,p-CREB expression,and GLUL expression as cytoplasmic GPER activated in CAFs,and a decreasing of c AMP production,p-PKA expression,p-CREB expression,and GLUL expression in CAFs treated with G15.Chromatin immunoprecipitation(Ch IP)assays showed that CREB could bind to the GLUL promoter.Knockdown of GPER or CREB significantly decreased the production of glutamine in CAFs and the conditional medium;(4)The tumor burden mice which were injected with MDA-MB-231 and engineered CAFs(CAF/sh GPER,CAF/sh GLUL,CAF/sh GPER+sh GLUL)had a significant small tumor,a slower growth and fewer lung metastases compared with the tumor burden mice injected with mixture of MDA-MB-231 and control CAFs.Conclusion: cytoplasmic GPER activated by estrogen enhances the expression of GLUL and LDHB via c AMP/PKA/CREB signaling pathway to facilitate glutamine synthesis and secretion.Glutamine,as a metabolic coupling mediator between CAFs(synthesis and secretion via GLUL and LDHB)and cancer cells(uptake and metabolism via ASCT2 and GLS1),promotes cancer cell proliferation,invasion,and et.al via fueling the mitochondrial activity in cancer cells.Part III:GPER in CAFs mediates the energy metabolism reprogramming to regulate immune escape of triple negative breast cancer in tumor microenvironmentObjective: This study aims to explore the molecular mechanism of GPER in cancer-associated fibroblasts(CAFs)from the tumor microenvironment in regulating immune escape of triple negative breast cancer(TNBC).Methods: Co-culture system of breast cancer cells with CAFs and breast cancer cells with Jurkat T cells,the function of GPER in CAFs was stimulated by E2,G1 or G15.The study employed cellular immunofluorescence(IF)and western blotting to detect PD-L1 expression and signaling pathway activation in TNBC cells.Detection of apoptosis in TNBC cells by breast cancer cell killing assay and flow cytometry.Results:(1)Treatment of tumor cells with E2/G1-stimulated CAFs cell culture supernatant led to increase the expression of PD-L1 in BT-549 or MDA-MB-231.Treatment of tumor cells with E2 and G15-stimulated CAFs cell culture supernatant led to decrease the expression of PD-L1 in BT-549 or MDA-MB-231.(2)Treatment of tumor cells with E2/G1-stimulated CAFs cell culture supernatant led to increase the expression of phosphorylated EGFR(p-EGFR),phosphorylated ERK(p-ERK),phosphorylated c-Jun(p-c-JUN),and PD-L1 in BT-549 and MDA-MB-231 cells.Treatment of CAFs with E2 and G15 rescued these changes.When tumor cells were co-cultured with Jurkat T cells,treatment with E2/G1-stimulated CAFs culture supernatant led to increase tumor cell survival and decrease apoptosis.E2 and G15-stimulated CAFs culture supernatant rescued these changes.(3)Treatment of tumor cells with E2-stimulated CAFs culture supernatant led to increase the expression of phosphorylation of related pathway proteins and PD-L1 in BT-549 or MDA-MB-231.As a result,tumor cells co-cultured with Jurkat T cells were more viable and exhibited less apoptosis.When tumor cells with sh ASCT2(glutamine transporter)were cultured with supernatant of E2-stimulated CAFs,the phosphorylation of related pathway proteins and PD-L1 expression in BT-549 and MDA-MB-231 cells decreased,leading to reduce viability and increase apoptosis of tumor cells co-cultured with Jurkat T cells.(4)Treatment of MDA-MB-231 and BT-549 cells with E2-stimulated CAFs cell culture supernatant led to increase the expression of phosphorylation of related pathway proteins and PD-L1 in BT-549 or MDA-MB-231.As a result,tumor cells co-cultured with Jurkat T cells were more viable and exhibited less apoptosis.When tumor cells were treated with AG126(25 μM)and cultured with supernatant of E2-stimulated CAFs,the phosphorylation of related pathway proteins and PD-L1 expression in BT-549 and MDA-MB-231 cells decreased,leading to reduce viability and increase apoptosis of tumor cells co-cultured with Jurkat T cells.Conclusion: Estrogen-activated cytoplasmic GPER in CAFs alters glutamine levels in the tumor microenvironment,and TNBC cells uptake glutamine from the microenvironment via the glutamine transporter(ASCT2).The uptake of glutamine from CAFs activates the intracellular EGFR/ERK/c-Jun signaling pathway,alters PD-L1 expression in tumor cells and affects the immune killing of TNBC cells.