Effects Of Cytoplasmic Gper Translocation Of Breast Cancer-Associated Fibroblasts On Tumor Energy Metabolism And Its Role In Tumor Multiple Drug Resistance

Estrogen plays a crucial role in breast tumor development.Traditionally, the estrogenic effects are mainly mediated by classic nuclear estrogen receptors(n ERs, ERα and ERβ) which are located in the nucleus,they are viewed as the transcription factors cis-acting with estrogen response element(ERE) to regulate the expression of downstream target genes. The G-protein coupled estrogen receptor(GPER) is identified as the third estrogen receptor which is functioned independently with n ERs in recent years. Both estrogen and estrogen receptor antagonist tamoxifen(TAM) can activate GPER to participate in numerous malignant biological properties in breast cancer, such as cell growth, cell invasion,pro-angiogenesis, drug resistance, and so on. Cancer-associated fibroblasts(CAFs) are the principal cellular component in the breast tumor microenvironment which secrete growth factors, cytokines, proteases, and hormones like estrogen through paracrine-dependent manner to the contribution of tumor growth, angiogenesis, extracellular matrix(ECM)remodeling, invasion, metastasis and chemoresistance. Thus, the cross-talk between tumor cells and CAFs is regarded as one of the pivotal steps inpromoting the development of breast cancer. Previous studies have obtained the following progress: GPER mainly located in the nucleus of CAFs when they are cultured alone and estrogen increases the target genes c-fos and CTGF expression through activating GPER/EGFR/ERK signaling pathway; TAM also activates GPER to enhance the expression of aromatizing enzyme gene(CYP19A1) and further increases the synthesis of regional estrogen that may play a key role in TAM resistance. However,the current researches are mainly focused on the GPER biological function of CAFs themselves, such as CAFs cell growth, cell migration and so on.Whether the cross-talk between breast cancer cells and CAFs affects GPER intracellular localization and its downstream biological effects in CAFs are still unknown. The present study intends to further explore the phenomenon and potent mechanism which breast cancer cells induce the cytoplasmic GPER translocation of CAFs and the possible effects of above GPER translocation on tumor energy metabolism and multiple drug resistance.The report was divided into the following five components:Section 1. Effects of breast cancer cells on GPER intracellular localization of CAFs and the correlation between GPER distribution of tumor stromal fibroblasts with the clinicopathological factors in breast cancerObjectives: To evaluate the effects of different breast cancer cellsubtypes on GPER intracellular localization of CAFs and the correlation between GPER distribution of tumor stromal fibroblasts with the clinicopathological factors in breast cancer.Methods: Immunohistochemistry was performed to detected the GPER expression and localization of tumor stromal fibroblasts(TSFs) in231 archival paraffin-embedded, formalin-fixed human primary breast tumor tissues, paired 21 normal mammary tissues, 53 paired TAM-sensitive(primary tumors, PTs) and TAM-resistant(metastasis tumors, MTs) breast tumor specimens. And GPER distribution of TSFs was correlated to clinicopathologic variables of tumors. Immunofluorescence was used to assess the change of GPER intracellular localization of CAFs after directly or indirectly co-culturing with tumor cells. Western blotting was utilized to explore the expression of cytoplasmic and nuclear GPER in CAFs under the treatment with diverse tumor cells conditioned medium(CM).Results:(1). 45% of primary breast tumor samples were identified to be GPER positive in stromal fibroblasts. 80 cases(80/104, 76.9%), which derived from ductal carcinoma in situ(DCIS), luminal A, luminal B, Her-2overexpression and triple-negative(TN) breast cancer subtypes, have mere cytoplasmic GPER-stained stromal fibroblasts. The other 24 cases(24/104,23.1%), which only derived from luminal A and luminal B subtypes, have combined cytoplasmic/nuclear GPER expression of TSFs. Compared with combined cytoplasmic/nuclear GPER expression pattern, the completecytoplasmic GPER expression in stromal fibroblasts was significantly associated with worse clinical stage(P=0.001). Moreover, the stromal fibroblasts with high level of cytoplasmic GPER were more likely identified in patients with Her-2 over-expressed, TNBC(P<0.001) and TAM-resistant breast cancer(P<0.05).(2). GPER was mainly located in the nucleus of CAFs and cytoplasm of breast cancer cells when cultured alone,which was inconsistent with the observed phenomenon in the stromal fibroblasts of breast tumor tissues. However, co-culturing CAFs with the above breast cancer cells after 24 h, a significant GPER translocation from the nucleus to the cytoplasm was observed in CAFs as detected in stromal fibroblasts of breast tumor. Condition medium(CM) from diverse tumor cells was used to treat CAFs at different time points(12h, 24 h, 36 h, 48h),the GPER cytoplasmic translocation in CAFs induced by tumor cells CM in a time-dependent manner was verified by Western blotting assay and immunofluorescence staining. More interestingly, the cytoplasmic GPER translocation in CAFs was more likely occurred under the treatment with CM from more malignant and drug(TAM or adriamycin) resistant breast tumor cells.Conclusion: GPER mainly located in the cytoplasm of stromal fibroblasts in breast cancer tissues. Breast cancer patients with absolute cytoplasmic GPER-expressing stromal fibroblasts might predict a poor prognosis or TAM-resistance in clinic, compared with combinedcytoplasmic/nuclear GPER expression pattern. The crosstalk between tumor cells and CAFs triggered the cytoplasmic GPER translocation in CAFs, this kind of translocation was likely to happen in high malignant and drug resistance breast cancer cells, indicating that the GPER cytoplasmic translocation in CAFs may present an important role in tumor progression and drug resistant.Section 2. The mechanism of breast cancer cells-induced GPER cytoplasmic translocation in CAFsObjectives: To investigate the mechanism of breast cancer cells-induced GPER cytoplasmic translocation in CAFs.Methods: Co-immunoprecipitation was performed to measure the mutual binding of GPER and nuclear export protein CRM1 in CAFs under the treatment with or without breast tumor cells CM. Bio-information assay was used to predict the potent nuclear export signals(NES) sequence in GPER amino acid sequence. Immunofluorescence was utilized to detect the roles of GPER NES sequence mutation on tumor cells CM-triggered GPER translocation in CAFs. Western blotting was used to determine the activation of MAPKs and PI3 K signaling pathway in CAFs treated with tumor cells CM. The effects of CRM1 specific inhibitor leptomycin B(LMB) and other specific inhibitors on the expression and distribution of GPER in CAFs treated with tumor cells CM were assessed by westernblotting and immunofluorescence, respectively.Results:(1). A significant mutual binding of GPER and CRM1 was tested in CAFs treated with tumor cells CM. A potent NES sequence which is similar with other steroid receptors was also identified by bio-information assay in GPER(YFINLAVADLILV). Mutation of the core NES sequences of GPER from LIL to AAA(YFINLAVADAAAV)dramatically reduced GPER export from nuclear to cytoplasm in CAFs co-cultured with tumor cells CM. This was further confirmed in CAFs under treatment of LMB(5ng/ml), a specific inhibitor of CRM1.(2). After exposure to tumor cells CM at diverse time points(5min, 15 min, 30 min,60min, 120min), a significantly phosphorylated ERK, AKT, P38 and JNK were detected in CAFs. However, treating CAFs with U0126(ERK inhibitor, 10μM), WM(PI3K inhibitor, 10μM), SB(P38 inhibitor, 10μM)and SP(JNK inhibitor, 10μM), separately, only PI3K/AKT signaling pathway specific inhibitor WM blocked the cytoplasmic GPER translocation of CAFs.Conclusion: CRM1, a nuclear export protein, mediated nuclear export of GPER via identified NES sequence in CAFs induced by breast cancer cells. Tumor cells CM-aroused PI3K/AKT signaling pathway, not MAPKs signaling pathways, was found to play a pivotal role in governing the CRM1-mediated cytoplasmic GPER translocation.Section 3. The activation of GPER-mediated downstream signaling pathway under the different GPER localization in CAFsObjectives: To evaluate the activation of GPER-mediated downstream signaling pathway under the different GPER localization in CAFs.Methods: Western blotting was used to test the effects of GPER agonists 17β-estradiol, G1 and TAM on the activation of GPER-mediated downstream EGFR/ERK, EGFR/AKT, c AMP/PKA signaling pathway in CAFs treated with breast cancer cells CM. Enzyme-linked immunosorbnent assay(ELISA) was performed to detect the change of intracellular c AMP expression under the diverse treatment condition. Western blotting and immunofluorescence were utilized to measure the activation of GPER/c AMP/PKA signaling downstream c AMP response element binding protein(CREB) and the expression of phosphorylated CREB in the nucleus of CAFs.Results:(1). E2(100n M), G1(100n M), and TAM(100n M)remarkably activated EGFR/ERK, EGFR/AKT signaling pathway when CAFs was cultured alone. Interestingly, we detected higher level of phosphorylated PKA(p-PKA), lower level of p-ERK, no much changed p-AKT and p-EGFR in CAFs co-cultured with MDA-MB-468/CM compared with CAFs alone. Consistently, more intracellular c AMPproduction which works as a biomarker of activated GPER/PKA signaling was also detected in CAFs co-cultured with MDA-MB-468/CM than that in CAFs alone. The increased c AMP and p-PKA levels induced by GPER agonists were notably blocked by LMB(10μM), WM(10μM) and GPER NES sequence mutation, which impeded the GPER export from nuclear to cytoplasm as proved above.(2). Activated GPER/c AMP/PKA by GPER agonists(E2, G1 and TAM) combined with MDA-MB-468/CM led to a notable increased nuclear p-CREB in CAFs, and the accumulated nuclear p-CREB was attenuated after inhibition of GPER/c AMP/PKA using c AMP specific inhibitor MDL-12330(20μM), PKA specific inhibitor H-89(1μM)or impeding cytoplasmic GPER translocation in CAFs.Conclusion: The cross-talk between breast cancer cells and CAFs not only affected the intracellular GPER localization of CAFs, but also further altered GPER-mediated downstream signaling pathway. Our data revealed that a novel cytoplasmic GPER-dependent GPER/c AMP/PKA pathway was activated in CAFs simulated by tumor cells. In addition, cytoplasmic GPER-dependent GPER/c AMP/PKA signaling was necessary for p-CREB expression and accumulation in nucleus of CAFs. Therefore, studying the GPER/c AMP/PKA/CREB pathway and its definite target genes would help to further enrich and understand the biological function of GPER in CAFs.Section 4. Effects of activated cytoplasmic GPER-dependent downstream c AMP/PKA/CREB signaling pathway on energy metabolism of CAFs in breast cancerObjectives: To investigate the effects of activated cytoplasmic GPER-dependent downstream c AMP/PKA/CREB signaling pathway on energy metabolism of CAFs.Methods: Bio-information assay was used to analyze the microarray data from reported researches to identify the potential target genes of transcription factor CREB. Quantitative real-time PCR(q RT-PCR) was performed to screen potent target genes of GPER/c AMP/PKA/CREB signaling pathway which was associated with glucose metabolism.Chromatin immunoprecipitation(CHIP) was utilized to detect the exact CREB downstream target genes. The glycolysis phenotype and mitochondrial activity of CAFs were tested by enzymes specific methods and Mito-Tracker Green probe, separately. 18F-fluorodeoxyglucose positron emission tomography/computed tomography(18F-FDG PET/CT)examination was performed to evaluate the effect of cytoplasmic stromal GPER on primary breast or metastatic tumor metabolic activity and its therapeutic response in clinic.Results:(1). Bio-information assay predicted that more than 1,400 CREB potential target genes in the database might involve in cellmetabolism. Among these targets, nine of interesting genes(including HK2,PFK1, PKM2, G6 PD, PDK4, PDK1, LDHA, LDHB, LDHC) encode the critical enzyme or kinase in glucose metabolism. Expressions of PDK4 and LDHB were enhanced under treatment of tumor cells CM combined with E2(100n M), G1(100n M) and TAM(100n M), and dramatically reduced by GPER antagonist G15(1μM) or H-89(1μM). Impeding GPER location in cytoplasm by GPER(M) or knocking down CREB with its specific si RNA decreased PDK4 and LDHB expression. Ch IP assay was further unveiled that E2, G1 and TAM induced the recruitment of CREB to PDK4 and LDHB promoter sequence.(2). In the co-culturing system of CAFs and tumor cells CM, the glucose consumption, lactate and pyruvate productions induced by GPER agonists were significantly depressed by H-89(1μM)and G15(1μM) in CAFs. The activated GPER/PKA signaling triggered by tumor cells CM and GPER agonists also impeded mitochondrial activity of CAFs. Moreover, the data from 18F-FDG PET/CT further present a strongly association between cytoplasmic GPER of stromal fibroblasts with tumor metabolic activity and clinical treatment.Conclusion: GPER agonists(E2, G1 and TAM) activates the cytoplasmic GPER-dependent downstream c AMP/PKA/CREB signaling pathway to regulate the target genes PDK4 and LDHB expression in CAFs,then the above process changed the metabolic pattern of CAFs which favored glycolysis to produce more lactate and pyruvate. The cytoplasmicGPER-mediated energy metabolism reprogramming(EMR) of CAFs might play a crucial role in altering tumor metabolic activity and drug resistance.Section 5. Effects of “CAFs-breast cancer cells” energy metabolic coupling triggered by GPER/PKA signaling of CAFs on tumor multiple drug resistanceObjectives: To explore the effects of “CAFs-breast cancer cells”energy metabolic coupling triggered by GPER/PKA signaling of CAFs on tumor multiple drug resistance.Methods: Mito-Tracker Green probe was used to detect the mitochondrial activity of breast cancer cells cultured with or without CAFs.Flow cytometry assays was performed to measure the effects of“CAFs-breast cancer cells” energy metabolic coupling(EMC) on cell-killing of clinical drugs including TAM, herceptin and epirubicin in breast cancer cells, further evaluation the protected function of EMC to tumor cells.Results:(1). The mitochondrial activities of tumor cells were significantly increased in co-cultured with E2(100n M)-treated CAFs,applying G15(1μM) or H-89(1μM) to CAFs could inhibit mitochondrial activities of tumor cells remarkably. Treating CAFs with CHC(monocarboxylate transporter inhibitor, 1m M), QUE(lactate transporter inhibitor, 10μM), or managing tumor cells with Met(mitochondrialcomplex I inhibitor, 100μM) and ATO(inhibitor of mitochondrial electron transport chain, 10μM) in the co-cultured system, the mitochondrial activities of tumor cells induced by EMC were notably reduced.(2). The E2-stimulated CAFs remarkably decreased MCF-7, SKBR3, and MDA-MB-468 cells drug-sensitivities to TAM, herceptin or epirubicin.Interestingly, the combination of Met and ATO with the clinical drugs was able to overcome the drug resistance of tumor cells co-cultured with CAFs.Conclusion: The activation of estrogen/GPER/PKA signaling triggered an aerobic glycolysis switch in CAFs. Then the glycolytic CAFs feed the extra pyruvate and lactate to tumor cells for their augment of mitochondrial activity, this energy metabolic-coupled “host-parasite relationship” between catabolic CAFs and anabolic cancer cells confers the tumor cells with multiple drug-resistant potential to conventional clinical treatments including endocrine therapy(tamoxifen), Her-2 targeted therapy(herceptin) and chemotherapy(epirubicin). Therefore, specially targeting GPER and its downstream signaling pathway in CAFs, and disturbing the EMC between CAFs and tumor cells might represent an effective way to improve the drug-sensitivity and reverse multiple drug resistance in clinical therapy of patients with breast cancer.