GPER‐regulated lncRNA‐Glu promotes glutamate secretion to enhance cellular invasion and metastasis in triple‐negative breast cancer

The FASEB JournalVolume 34, Issue 3 p. 4557-4572 RESEARCH ARTICLEFull Access GPER-regulated lncRNA-Glu promotes glutamate secretion to enhance cellular invasion and metastasis in triple-negative breast cancer Jiali Yin, Jiali Yin Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, Chongqing, ChinaSearch for more papers by this authorGang Tu, Gang Tu Department of Endocrine and Breast Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, ChinaSearch for more papers by this authorMeixi Peng, Meixi Peng Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, Chongqing, ChinaSearch for more papers by this authorHuan Zeng, Huan Zeng Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, Chongqing, ChinaSearch for more papers by this authorXueying Wan, Xueying Wan Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, Chongqing, ChinaSearch for more papers by this authorYina Qiao, Yina Qiao Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, Chongqing, ChinaSearch for more papers by this authorYilu Qin, Yilu Qin Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, Chongqing, ChinaSearch for more papers by this authorManran Liu, Corresponding Author Manran Liu manranliu@cqmu.edu.cn Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, Chongqing, China Correspondence Manran Liu, Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, #1 Yi-Xue-Yuan Rd., Yu-zhong District, Chongqing 400016, China. Email: manranliu@cqmu.edu.cn Haojun Luo, Department of Thyroid and Breast Surgery, The Second Affiliated Hospital of Chongqing Medical University, #74 Lin-Jiang Rd., Yu-zhong District, Chongqing 400010, China. Email: luohaojun@hospital.cqmu.edu.cnSearch for more papers by this authorHaojun Luo, Corresponding Author Haojun Luo luohaojun@hospital.cqmu.edu.cn Department of Thyroid and Breast Surgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China Correspondence Manran Liu, Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, #1 Yi-Xue-Yuan Rd., Yu-zhong District, Chongqing 400016, China. Email: manranliu@cqmu.edu.cn Haojun Luo, Department of Thyroid and Breast Surgery, The Second Affiliated Hospital of Chongqing Medical University, #74 Lin-Jiang Rd., Yu-zhong District, Chongqing 400010, China. Email: luohaojun@hospital.cqmu.edu.cnSearch for more papers by this author Jiali Yin, Jiali Yin Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, Chongqing, ChinaSearch for more papers by this authorGang Tu, Gang Tu Department of Endocrine and Breast Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, ChinaSearch for more papers by this authorMeixi Peng, Meixi Peng Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, Chongqing, ChinaSearch for more papers by this authorHuan Zeng, Huan Zeng Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, Chongqing, ChinaSearch for more papers by this authorXueying Wan, Xueying Wan Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, Chongqing, ChinaSearch for more papers by this authorYina Qiao, Yina Qiao Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, Chongqing, ChinaSearch for more papers by this authorYilu Qin, Yilu Qin Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, Chongqing, ChinaSearch for more papers by this authorManran Liu, Corresponding Author Manran Liu manranliu@cqmu.edu.cn Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, Chongqing, China Correspondence Manran Liu, Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, #1 Yi-Xue-Yuan Rd., Yu-zhong District, Chongqing 400016, China. Email: manranliu@cqmu.edu.cn Haojun Luo, Department of Thyroid and Breast Surgery, The Second Affiliated Hospital of Chongqing Medical University, #74 Lin-Jiang Rd., Yu-zhong District, Chongqing 400010, China. Email: luohaojun@hospital.cqmu.edu.cnSearch for more papers by this authorHaojun Luo, Corresponding Author Haojun Luo luohaojun@hospital.cqmu.edu.cn Department of Thyroid and Breast Surgery, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, China Correspondence Manran Liu, Key Laboratory of Laboratory Medical Diagnostics designated by Chinese Ministry of Education, Chongqing Medical University, #1 Yi-Xue-Yuan Rd., Yu-zhong District, Chongqing 400016, China. Email: manranliu@cqmu.edu.cn Haojun Luo, Department of Thyroid and Breast Surgery, The Second Affiliated Hospital of Chongqing Medical University, #74 Lin-Jiang Rd., Yu-zhong District, Chongqing 400010, China. Email: luohaojun@hospital.cqmu.edu.cnSearch for more papers by this author First published: 06 February 2020 https://doi.org/10.1096/fj.201901384RRCitations: 2 [Correction added on February 13, 2020, after first online publication: Updated version of Figure 6B has been replaced.] AboutFiguresReferencesRelatedInformationPDFSectionsAbstract1 INTRODUCTION2 MATERIALS AND METHODS3 RESULTS4 DISCUSSION ACKNOWLEDGMENTS CONFLICT OF INTEREST AUTHOR CONTRIBUTIONSSupporting InformationREFERENCESCiting LiteraturePDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessClose modalShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract Triple-negative breast cancer (TNBC) is a group of breast cancer with heterogeneity and poor prognosis and effective therapeutic targets are not available currently. TNBC has been recognized as estrogen-independent breast cancer, while the novel estrogen receptor, namely G protein-coupled estrogen receptor (GPER), was claimed to mediate estrogenic actions in TNBC tissues and cell lines. Through mRNA microarrays, lncRNA microarrays, and bioinformatics analysis, we found that GPER is activated by 17β-estradiol (E2) and GPER-specific agonist G1, which downregulates a novel lncRNA (termed as lncRNA-Glu). LncRNA-Glu can inhibit glutamate transport activity and transcriptional activity of its target gene VGLUT2 via specific binding. GPER-mediated reduction of lncRNA-Glu promotes glutamate transport activity and transcriptional activity of VGLUT2. Furthermore, GPER-mediated activation of cAMP-PKA signaling contributes to glutamate secretion. LncRNA-Glu-VGLUT2 signaling synergizes with cAMP-PKA signaling to increase autologous glutamate secretion in TNBC cells, which activates glutamate N-methyl-D-aspartate receptor (NMDAR) and its downstream CaMK and MEK-MAPK pathways, thus enhancing cellular invasion and metastasis in vitro and in vivo. Our data provide new insights into GPER-mediated glutamate secretion and its downstream signaling NMDAR-CaMK/MEK-MAPK during TNBC invasion. The mechanisms we discovered may provide new targets for clinical therapy of TNBC. Abbreviations CaMK Ca2+/calmodulin kinase E2 17β-estradiol EGFR epidermal growth factor receptor ERα estrogen receptor alpha GPER G protein-coupled estrogen receptor HER2 human epidermal growth factor receptor 2 NMDAR N-methyl-D-aspartate receptor PR progesterone receptor TNBC triple-negative breast cancer VGLUT2 vesicular glutamate transporter 2 1 INTRODUCTION Breast cancers characterized by negative expression of estrogen receptor alpha (ERα), progesterone receptor (PR), and lack of amplification of human epidermal growth factor receptor 2 (HER2) are classified as triple-negative breast cancer (TNBC), a subtype of breast cancers.1 This subtype represents 15% of all breast cancers and exhibits the most aggressive behavior among the four known subtypes of breast cancer.2 Compared with other subtypes of breast cancer, TNBC is more prone to form distant metastasis,3 especially in the lungs and brain,4 both of which eventually lead to breast cancer-specific death in short-term. Concerning the systemic therapy, there has been no available target in TNBC for a long time. Thus, blocking TNBC metastasis should be the most effective strategy to improve patients' outcomes. Estrogens are essential etiological factors for breast cancer,5 and endocrine therapy is crucial for the multidisciplinary management of breast cancer patients. However, estrogen carcinogenesis was long disregarded in TNBC, logically because of the absence of ERα. The identification of an alternative ER, the G protein-coupled estrogen receptor (GPER), which was identified as a membrane-associated receptor mediating rapid and nongenomic estrogenic effects, including transactivation of epidermal growth factor receptor (EGFR) and production of second messengers such as cAMP, calcium, and inositol triphosphate,6, 7 have challenged the traditional concept that TNBC is estrogen-independent. In our and other early reports, GPER was detected in about 60% of primary TNBC samples,8, 9 as well as in several TNBC cell lines. Besides, GPER was triggered by ligands such as 17β-estradiol (E2) and bisphenol A, resulting in promotion of proliferation and migration of TNBC cells.10, 11 Considering the effectiveness, convenience, and safety of endocrine therapy, GPER was included as a candidate biomarker and a potential therapeutic target for TNBC.12 However, its role in TNBC metastasis remains unclear, and further evaluation is urgently needed. Although the transcriptional effects of estrogens were attributed to nuclear ERα/β, increasing evidence supported that activation of GPER could be involved in extensive transcriptional regulation.13 Microarray strategies were repeatedly applied in the GPER-related study, leading to the identification of a broad spectrum of GPER-targeted genes in different cell backgrounds.14-16 However, a comprehensive perspective of transcriptional actions triggered by GPER was not evident in TNBC cells. Moreover, long non-coding RNA (lncRNA) is an important subgroup of non-coding RNA that could affect cellular biological behaviors by regulating gene expression and function.17 A growing number of studies suggest that lncRNAs are involved in the progression of breast cancer.18 Besides, estrogen also reportedly induces a set of related lncRNAs, which bear prognosis value for clinical outcomes of breast cancer patients.19, 20 Therefore, there seems to be a lot of unknown events involving lncRNAs underlying the known transcriptional effects of estrogen in TNBC. In oncology, cellular metabolism significantly differs from normal conditions and largely contributes to cancer initiation and development.21 Glutamate is a pivotal compound in physiological and pathophysiological cellular metabolism. Increased glutamate was reported to stimulate migration and invasion of several types of cancer cells.22 Glutamate could trigger several downstream pathways through N-methyl-D-aspartate receptor (NMDAR), such as GKAP signaling to promote invasion of cancer cells.23 Although a higher concentration of secreted glutamate was observed in TNBC cells as compared with ERα- and HER2-positive breast cancer cells,24 the mechanism of increased glutamate secretion still requires further investigation. 2 MATERIALS AND METHODS Cell culture, shRNA, and siRNA Human TNBC cell line MDA-MB-468 was obtained from ATCC (Manassas, VA, USA) and HCC1806 was obtained from Kunming Cell Bank of the Chinese Academy of Sciences (Kunming, China). MDA-MB-468 and HCC1806 cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% of FBS (Gibco). All cells were changed to serum- and phenol-free RPMI-1640 medium for 24 hours before treatment. The lentivirus expression vector and its infective lentivirus were acquired from GenePharma (Shanghai, China). MDA-MB-468 and HCC1806 cells with stably overexpressed lncRNA-Glu or silenced lncRNA-Glu and their control cells were constructed to be engineered cells by lentivirus infection and screening with puromycin. The sequence of negative control shRNA is 5'-TTCTCCGAACGTGTCACGT-3'. GPER target sequences are shRNA #1:5'-AGTACGTGATCGGCCTGTT-3' and shRNA #2:5'-CGCTCCCTGCAAGCAGTCTTT-3'. VGLUT2 target sequences are shRNA #1:5'- CTATGGTGGAGTTATATTT-3' and shRNA #2:5'-TGAAACCAGAGATAGCAAATC-3'. The sequences of lncRNA-Glu siRNA are siRNA #1:5'-TTCAAAACCGGGAATTTACAT-3' and siRNA #2:5'-AATAAGTCTTCAAAACCGGGA-3'. The sequences of NR2b siRNA are siRNA #1:5'-CCAGTTTGGCCCATCAATT-3' and siRNA #2:5'-GTGACTACATCAGTGAGGT-3'. Quantitative real-time PCR and western blotting Total RNA from cells or tissues was extracted using RNAiso Plus (Takara, Japan). The RNA was converted into cDNA using a PrimeScript RT reagent Kit (Takara, Japan) according to the manufacturer instructions. Real-time PCR was conducted using SYBR Premix Ex TaqTM II (Takara). Each experiment was conducted in triplicates. Gene expression was calculated with β-Actin as an endogenous control using the comparative 2−ΔΔCT method. The sequences of primers (Sangon Biotech, Shanghai, China) used were as follows: GPER forward (5'-ACGAGACTGTGAAATDCGCAACCA-3'), GPER reverse (5'-ATCAGGCTGGAGGTGCACTTGGAA-3'); VGLUT2 forward (5'-CATCCATGCCAGTCTATGCAA-3'), VGLUT2 reverse (5'-ATGCTGGCTGACTAATAAGCAA-3'); MMP7 forward (5'-GCTACAGTGGGAACAGGCTC-3'), MMP7 reverse (5'-GGGATCTCTTTGCCCCACAT-3'); lncRNA-Glu forward (5'-TATCACAGAAGCCTTGGGAA-3'), lncRNA-Glu reverse (5'-TTTTGAATATGCTCTGCTCC-3'). The total proteins were obtained using RIPA protein extraction buffer (Boster, China) with protease inhibitor (Beyotime, Shanghai, China) and separated using SDS-PAGE. The specific primary antibody against the indicated protein was incubated at 4°C overnight. Next, membranes were incubated with horseradish peroxidase-conjugated secondary antibody. The indicated proteins were visualized using the enhanced chemiluminescence system (Amersham, Freiburg, Germany). Reagents 17β-estradiol (E2), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The GPER-specific agonist G1 and antagonist G36 were obtained from Tocris (Ellisville, MO, USA). The cAMP inhibitor MDL-12,330 (MDL) was purchased from Santa Cruz Biotechnology, Inc (Dallas, TX, USA). The cAMP agonist forskolin (FSK), and the PKA inhibitor H-89 were purchased from Beyotime (Shanghai, China). The NMDAR inhibitor MK801 was purchased from Sigma-Aldrich (St. Louis, MO, USA). E2 was dissolved in ethanol, while other reagents were dissolved in DMSO. The antibodies applied in western blotting are as follows: GPER (1:250, ab39742, Abcam, Cambridge, MA, USA), p-NR2b (1:1000, ab193286, Abcam), p-PKA (1:1000, BS4345, Bioworld, St. Louis Park, MN,USA), VGLUT2 (1:1000, #71555, Cell Signaling Technology, Danvers, MA, USA), p-CaMKII (1:1000, #12716, CST), CaMKII (1:1000, #3362, CST), p-MEK (1:500, #9154, CST), MEK (1:1000, #4694, CST), p-MAPK (1:1000, #9101, CST), MAPK (1:1000, #9102, CST), p-CREB (1:500, #9198, CST), CREB (1:1000, #9197, CST), and β-Actin (1:1000, ZSBIO, Beijing, China). Analyses of microarrays results More than 18 000 mRNAs and 60 000 lncRNAs were detected using Agilent Human mRNA and lncRNA microarrays, respectively. We decided that mRNAs were potential GPER-regulated mRNAs based on the following criteria: more than 1.5-fold induction by both E2 and G1; more than 1.5-fold reduction of the E2/G1 response by G36 and GPER knockdown. These criteria identified 589 potential GPER-regulated mRNAs. LncRNAs were considered to be potential GPER-regulated lncRNAs by the following criteria: more than 2-fold induction by both E2 and G1; more than 2-fold reduction of the E2 response by G36. These criteria identified 2102 potential GPER-regulated lncRNAs. Clinical samples In total, 96 paired human breast tumor tissues and adjacent normal counterparts were collected from TNBC patients undergoing surgery in the First Affiliated Hospital of Chongqing Medical University. All enrolled patients consented to participate in this study and none of them received radiotherapy or chemotherapy before surgery. The clinical samples were stored in liquid nitrogen immediately after surgery. Our study design was approved by the Ethics Committee of Chongqing Medical University. Immunohistochemistry analyses and scoring Immunohistochemical staining was conducted as previously described.25 The results of immunohistochemical staining were analyzed quantitatively according to the proportion of positive cells and the intensity of staining as previously described.26 Proportion scores were assigned as follows: 0 if 0% of the TNBC cells showed positive staining, 1 if 0%-1%, 2 if 1%-10%, 3 if 11%-30%, 4 if 31%-70%, and 5 if 71%-100%. The intensity of staining was rated as follows: 0 for negative, 1 for weak, 2 for moderate, and 3 for strong. Proportion scores and the intensity of staining were combined to get a total score (range of 0-8), and the total score equal to or greater than 4 was considered as positive expression. Cell invasion assay The HCC1806 and MDA-MB-468 cells were used in cell invasion assays, which were conducted as previously described.25 Briefly, cells were counted, and 8 × 103 cells were suspended in 200 µL of serum-free medium. Next, cells were seeded into Boyden chambers (Millipore, Darmstadt, Germany) of 8µm-pore membranes coated with Matrigel (1:7.5) (Corning BioCoat, Bedford, OH, USA). The cells were then treated with MDL (20 µM), H-89 (1 μM), E2 (100 nM), G1 (100 nM). After incubation for 18 hours, the invasive cells were stained with 0.5% crystal violet and counted by Nikon Eclipse 80i microscope (Nikon, Japan) in five randomly selected views. All experiments were conducted three times repeatedly and independently (means ± SD). In vivo assays for metastasis Forty-five 4-week old female nude mice were purchased from HFK BIOSCIENCE (Beijing, China). Mice acclimated for one week before experiments. MDA-MB-468 cells (5 × 106) in 200 µL of PBS: Matrigel at a ratio of 1:1 were injected into the mammary fat pad of nude mice. Once tumor sizes reached 10-30 mm3, the mice were divided into nine groups (n = 5) and injected intraperitoneally (ip) with E2 [20 µg/kg body weight (b.w.)], G1 (5 mg/kg b.w.), H-89 + G1 (H-89 10 mg/kg b.w. + G1 5 mg/kg b.w.), MK801 + G1 (MK801 1mg/kg b.w. + G1 5 mg/kg b.w.) two or three times a week for 4 weeks, respectively. The exogenous supplementation of glutamate in the drinking water for mice was set to 1 mM. The mouse lungs were harvested, sectioned into cryosections (5 μm), and stained with hematoxylin and eosin (H&E, Baso, Zhuhai, China) for histological assessment. The metastatic index was obtained by the number and diameter of metastatic foci of lung surface.27 The metastatic area per lung section was calculated using the software ImageJ. RNA pull-down assay Using the biotin RNA labeling mix and T7 RNA polymerase, lncRNA-Glu was transcribed in vitro according to the manufacturer's instruction (Invitrogen, New York, USA). Then RNA pull-down assay was conducted by Pierce Magnetic RNA-Protein Pull-Down Kit (ThermoFisher Scientific, Waltham, MA USA). Briefly, 40μl of streptavidin magnetic beads were used to pull-down the biotinylated RNA and 80μg of protein to bind RNA. Finally, the RNA-binding protein was washed, and the retrieved protein was analyzed by western blotting. RNA immunoprecipitation assay The RNA immunoprecipitation (RIP) assay was conducted by EZ-Magna RIP RNA-Binding Protein Immunoprecipitation Kit, according to the manufacturer's instruction (Millipore, USA). Cells were cultured in 10 cm plates to 80% confluence. Cells were suspended in 100 µL of cell lysate. Then, 5 µg of purified antibodies and corresponding IgG were added to cell lysate. The mixture was incubated with rotation at 4°C overnight. The immunoprecipitated RNAs were analyzed by quantitative real-time PCR (qRT-PCR). Anti-GAPDH antibody (1:200, ab9485, Abcam) and normal rabbit IgG (Cat. #PP64B, Millipore) were used as controls. Preparation of membrane fractions and glutamate uptake For the glutamate transport assay of vesicle membranes, 100 µg of vesicular membrane proteins in uptake buffer were mixed with 4 mM ATP, 4 mM KCl, and 50 µM L-[3H]glutamate (potassium salt, 0.4Ci/mmol, Amersham Pharmacia Biotech) at 25°C for different times. Uptake was stopped by the addition of 4 × 2 mL of ice-cold 0.15M KCl and immediate filtration. Radioactivity was detected with a liquid scintillation spectrophotometer. Measurement of glutamate concentration Glutamate levels in cell-culture media and xenograft tissues were detected by Glutamate Assay Kit according to the manufacturer's instruction (ab83389, Abcam) and using the standard curve obtained. Statistical analysis Experimental data are expressed as means ± SD of at least three independent experiments. All statistical analyses were done using the SPSS standard version 20 software. The lncRNA-Glu RNA levels in human samples were analyzed using the paired t test. The lncRNA-Glu level, luciferase activity, and mRNA level were analyzed using two-way analysis of variance. The Chi-square test or Fisher's exact test was used for calculating the correlation between the analyzed GPER, VGLUT2, NR2b expression, and clinicopathological factors. The Chi-square test was used for evaluating the correlation between VGLUT2, NR2b expression, and GPER expression. p values less than .05 were considered statistically significant. 3 RESULTS GPER mediates changes of mRNA and lncRNA profiles in TNBC Our previous study has shown that GPER is involved in TNBC malignancy.25 Since CTGF (connective tissue growth factor) has been identified as a GPER target gene, we verified the effectiveness of GPER agonists E2 and G1 and GPER antagonist G36 treatments by detecting expression of CTGF mRNA (Figure S1A). To clarify the changes of GPER-mediated mRNA and lncRNA expressions in TNBC cells, TNBC cells with a high level of GPER, MDA-MB-468, and their GPER-knocked down cells (Figure S1B,C) were employed and treated with or without E2 (100 nM), G1 (100 nM), and G36 (100 nM, combined with E2 (100 nM)) for one hour, respectively, before undergoing mRNA and lncRNA profiles analyses using Agilent Human mRNA and lncRNA microarrays. We conducted analyses of mRNA and lncRNA microarrays results. In total, 589 mRNAs were induced by both E2 and G1 while reduced by G36 and GPER knockdown. For lncRNAs, 2102 were induced by both E2 and G1 while reduced by G36. We identified these 589 mRNAs as potential GPER-regulated mRNAs (Figure 1A) and 2102 lncRNAs as potential GPER-regulated lncRNAs (Figure 1B). To validate these mRNAs or lncRNAs scanned by microarrays, 10 randomly chosen mRNAs (Figure1C) and lncRNAs (Figure 1D) were detected by qRT-PCR. Figure 1Open in figure viewer Analysis of mRNA and lncRNA microarray results. A, Venn diagram of mRNAs regulated at least 1.5-fold by any one of the treatments. Of these mRNAs, 589 were considered to be potential GPER-regulated mRNAs by the criteria. B, Venn diagram of lncRNAs regulated at least 2-fold by any one of the treatments. Of these lncRNAs, 2102 were considered to be potential GPER-regulated lncRNAs by the criteria. C and D, Ten of dysregulated mRNAs (C) and lncRNAs (D) assessed by Agilent Human microarray were detected by qRT-PCR. Data are shown as fold change between G1 treated control and GPER-knocked down cells of MDA-MB-468 and HCC1806. E and F, The major changed pathways (P < .05) of 589 mRNAs (E) and predicted target genes of 2102 lncRNAs (F) were enriched by DAVID v.6.8. The black column represents the number of genes/predicted genes located in the pathway. Representative genes and the gene of interest were shown on the right panel. Percentage (%) = altered genes/total genes in a signaling pathway. G, Heatmap of VGLUT2 levels in treated and untreated samples assessed by mRNA microarray. For each condition, the color indicates the ratio of the values. GPER KD, GPER knockdown In order to understand the correlative signaling pathways, we tried to predict the target genes of the 2102 lncRNAs using the algorithms based on cis- and trans-regulatory mechanisms of lncRNAs28 and obtained 625 target genes of GPER-regulated lncRNAs. These 625 target genes and the identified 589 mRNAs were classified by DAVID v.6.8 using KEGG pathway database. Several well-known canonical signaling pathways, such as cAMP, NF-kappa B, TNF, PI3K-Akt, and Wnt, which are related to cancer cell invasion, were identified in both the mRNA and the lncRNA datasets (Figure 1E,F). Interestingly, glutamatergic synapse pathway, which is associated with tumor growth and invasion,29 aroused our attention among these signaling pathways. Moreover, vesicular glutamate transporter 2 (VGLUT2), which exports glutamate in neurons,30 was found to be located on the glutamatergic synapse pathway. Also, VGLUT2 was induced by E2 and G1 while reduced by G36 and GPER depletion, according to the mRNA microarray (Figure 1G). These findings indicate that glutamatergic synapse pathway, especially VGLUT2, may contribute to GPER-mediated breast tumor metastasis by exporting glutamate. VGLUT2 upregulated by activated GPER increased glutamate secretion To demonstrate VGLUT2 expression in TNBC tissues, we evaluated mRNA levels of VGLUT2 in 96 paired TNBC samples and adjacent normal tissues using qRT-PCR and 69.8% (67/96) of tumors showed increased expression of VGLUT2 compared with coupled normal tissues (Figure 2A). By Immunohistochemistry (IHC) test, we confirmed the positive expression of VGLUT2 in 55.2% samples of this series. Interestingly, VGLUT2 expression paralleled with GPER expression in most cases (Figure 2B, Table S1), indicating a positive link between the expression of VGLUT2 and GPER in TNBC tissues. Indeed, the significantly positive correlation between VGLUT2 and GPER was further confirmed by IHC scores in TNBC tissues (Figure 2C). Besides, VGLUT2 expression was significantly correlated with lymph node status and clinical latter staging in the patients (Table S1). Figure 2Open in figure viewer VGLUT2 upregulated by activated GPER increased glutamate secretion. A, Fold change in VGLUT2 expression between TNBC tissues (n = 96) and normal tissues (n = 96) normalized to β-Actin expression using qRT-PCR. B, Representative images of GPER and VGLUT2 expression from the same sample of two TNBC patients by IHC. Scale bars, 100 μm. C, The correlation between GPER and VGLUT2 expression was analyzed (Pearson product moment correlation test). D, VGLUT2 expression in MDA-MB-468, HCC1806, and corresponding GPER knockdown cells treated with E2 (100 nM), G1 (100 nM), and G36 (100 nM) combined with E2 and G1 by western blotting analysis. E, GPER agonists increased glutamate concentration in the medium of MDA-MB-468 and HCC1806 cells. Data are shown as means ± SD of three independent experiments, each with similar trends (**P < .01) Furthermore, the levels of VGLUT2 were increased in TNBC cells MDA-MB-468 and HCC1806 under treatment with E2 and G1, respectively; G36 treatment could efficiently abolish E2 and G1 stimulation to VGLUT2 expressions; GPER depletion in MDA-MB-468 and HCC1806 cells (Figure S1D,E) canceled the stimulation of E2 and G1 to GPER-mediated VGLUT2 expression (Figure 2D). Besides, increased glutamate concentrations were detected in the supernatant of MDA-MB-468 and HCC1806 cells stimulated with E2 and G1; and decreased by G36; consistently, GPER depletion blunted glutamate secretion induced by treatment of E2 and G1 (Figure 2E). These data indicate that GPER activation increases VGLUT2 expression and consequently levels of extracellular glutamate in TNBC cells. Activated GPER downregulates lncRNA-Glu to increase glutamate transport activity and transcriptional activity of VGLUT2 As VGLUT2 was predicted to be the target gene of lncRNA-Glu (glutamate signaling associated lncRNA), we wondered whether lncRNA-Glu takes part in GPER-VGLUT2-glutamate signaling. We measured the expression of lncRNA-Glu by qRT-PCR and found that in TNBC tissues, lncRNA-Glu expression and GPER scores were significantly inversely correlated (Figure 3A). To understand the relationship between GPER and lncRNA-Glu, TNBC cells MDA-MB-468 and HCC1806 were employed. Treatment of E2 and G1 to MDA-MB-468 and HCC1806 resulted in reduced lncRNA-Glu, whereas G36 treatment could antagonize the E2 and G1 induced decrease of lncRNA-Glu. Consistently, GPER depletion canceled the E2 and G1 induced downregulation of lncRNA-Glu in MDA-MB-468 and HCC1806 cells (Figure 3B). These data suggest that lncRNA-Glu is a GPER-related lncRNA in TNBC cells. Figure 3Open in figure viewer Downregulated lncRNA-Glu increases glutamate transport activity and transcriptional activity of VGLUT2. A, T