Oxidized ATM governs stemness of breast cancer stem cell through regulating ubiquitylation and acetylation switch.

Cancer stem cells (CSCs), as parts of tumor initiation cells, play a crucial role to tumorigenesis, development and recurrence. However, the complicated mechanisms of CSCs to adapt to tumor microenvironment and its stemness maintenance remains unclear. Here, we show that oxidized ATM, a hypoxia-activated cytoplasm ATM, acts a novel function to maintain CSC stemness in triple-negative breast cancer cells (BCSCs) via regulating histone H4 acetylation. Mechanistically, oxidized ATM phosphorylates TRIM21 (a E3 ubiquitin ligase) serine 80 and serine 469. Serine 80 phosphorylation of TRIM21 is essential for the ubiquitination activity of TRIM21. TRIM21 binds with SIRT1 (one of deacetylase), resulting in ubiquitylation-mediated degradation of SIRT1. The reduced SIRT1 leads to increase of histone H4 acetylation, thus facilitating CSC-related gene expression. Clinical data verify that high level of ATM in breast tumors is positively correlated with malignant grade, and is closely related with low SIRT1, high p-TRIM21, and high CD44 expression. In conclusion, our study provides a novel mechanism by which oxidized ATM governing BCSCs stemness and reveals an important link among oxidized ATM, histone acetylation, and BCSCs maintenance.

LAPTM4B promotes AML progression through regulating RPS9/STAT3 axis.

Acute myeloid leukemia (AML) is a heterogeneous disorder with high morbidity and mortality under the existing treatment strategy. Here, we found that lysosome-associated protein transmembrane 4 beta (LAPTM4B) was frequently upregulated in AML, and high LAPTM4B was associated with poor outcome. Moreover, LAPTM4B promoted leukemia progression in vitro and in vivo. Mechanically, LAPTM4B interacted with RPS9, and positively regulated RPS9 protein stability, which enhanced leukemia cell progression via activating STAT3. Our findings indicate for the first time that LAPTM4B contributes to leukemia progression in a RPS9/STAT3-dependent manner, suggesting that LAPTM4B may serve as a promising target for treatment of AML.

Cytoplasmic Expression of TP53INP2 Modulated by Demethylase FTO and Mutant NPM1 Promotes Autophagy in Leukemia Cells.

Acute myeloid leukemia (AML) with a nucleophosmin 1 (NPM1) mutation is a unique subtype of adult leukemia. Recent studies show that NPM1-mutated AML has high autophagy activity. However, the mechanism for upholding the high autophagic level is still not fully elucidated. In this study, we first identified that tumor protein p53 inducible nuclear protein 2 (TP53INP2) was highly expressed and cytoplasmically localized in NPM1-mutated AML cells. Subsequent data showed that the expression of TP53INP2 was upregulated by fat mass and obesity-associated protein (FTO)-mediated m6A modification. Meanwhile, TP53INP2 was delocalized to the cytoplasm by interacting with NPM1 mutants. Functionally, cytoplasmic TP53INP2 enhanced autophagy activity by promoting the interaction of microtubule-associated protein 1 light chain 3 (LC3) - autophagy-related 7 (ATG7) and further facilitated the survival of leukemia cells. Taken together, our study indicates that TP53INP2 plays an oncogenic role in maintaining the high autophagy activity of NPM1-mutated AML and provides further insight into autophagy-targeted therapy of this leukemia subtype.

Targeted activation of GPER enhances the efficacy of venetoclax by boosting leukemic pyroptosis and CD8+ T cell immune function in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a rapidly progressing and often fatal hematopoietic malignancy. Venetoclax (VEN), a recent FDA-approved BCL-2 selective inhibitor, has high initial response rates in elderly AML patients, but the majority of patients eventually acquire resistance. Multiple studies have demonstrated that the female sex is associated with better outcomes in patients with AML, which are predominantly attributed to estrogen signaling. As a novel membrane estrogen receptor, G protein-coupled estrogen receptor (GPER)-mediated-rapid estrogen effects have attracted considerable attention. However, whether targeting GPER enhances the antileukemic activity of VEN is unknown. In this study, we first demonstrated that GPER expression was dramatically reduced in AML cells owing to promoter hypermethylation. Furthermore, pharmacological activation of GPER by G-1 combined with VEN resulted in synergistic antileukemic activity in vitro and in vivo. Mechanistically, G-1/VEN combination synergistically triggered concurrent mitochondria-related apoptosis and gasdermin E (GSDME)-dependent pyroptosis by activating p38-MAPK/myeloid cell leukemia 1 (MCL-1) axis. Importantly, leukemic pyroptosis heightened CD8+ T cell immune function by releasing interleukin (IL)-1ß/18 into the tumor microenvironment. Our study corroborates that GPER activation shows a synergistic antileukemic effect with VEN, making it a promising therapeutic regimen for AML.

Circulating plasma exosomal long non-coding RNAs LINC00265, LINC00467, UCA1, and SNHG1 as biomarkers for diagnosis and treatment monitoring of acute myeloid leukemia.

Exosomal long non-coding RNAs (lncRNAs) have emerged as a cell-free biomarker for clinical evaluation of cancers. However, the potential clinical applications of exosomal lncRNAs in acute myeloid leukemia (AML) remain unclear. Herein, we attempted to identify plasma exosomal lncRNAs as prospective biomarkers for AML. In this study, plasma exosomes were first successfully extracted from AML patients and healthy donors (HD). Subsequently, the downregulated plasma exosomal lncRNAs (LINC00265, LINC00467, and UCA1) and the upregulated plasma exosomal lncRNA (SNHG1) were identified in AML patients (n=65) compared to HD (n=20). Notably, individual exosomal LINC00265, LINC00467, UCA1, or SNHG1 had a capability for discriminating AML patients from HD, and their combination displayed better efficiency. Furthermore, exosomal LINC00265 and LINC00467 were increased expressed in patients achieving complete remission after chemotherapy. Importantly, there was upregulation of exosomal LINC00265 and downregulation of exosomal SNHG1 upon allogeneic hematopoietic stem cell transplantation. Additionally, these lncRNAs were high stability in plasma exosomes. Exosomal LINC00265, LINC00467, UCA1, and SNHG1 may act as promising cell-free biomarkers for AML diagnosis and treatment monitoring and provide a new frontier of liquid biopsy for this type of cancer.

Targeting Mitochondrial Oxidative Phosphorylation Eradicates Acute Myeloid Leukemic Stem Cells.

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by multiple cytogenetic and molecular abnormalities, with a very poor prognosis. Current treatments for AML often fail to eliminate leukemic stem cells (LSCs), which perpetuate the disease. LSCs exhibit a unique metabolic profile, especially dependent on oxidative phosphorylation (OXPHOS) for energy production. Whereas, normal hematopoietic stem cells (HSCs) and leukemic blasts rely on glycolysis for adenosine triphosphate (ATP) production. Thus, understanding the regulation of OXPHOS in LSCs may offer effective targets for developing clinical therapies in AML. This review summarizes these studies with a focus on the regulation of the electron transport chain (ETC) and tricarboxylic acid (TCA) cycle in OXPHOS and discusses potential therapies for eliminating LSCs.

Mutant NPM1-Regulated FTO-Mediated m6A Demethylation Promotes Leukemic Cell Survival via PDGFRB/ERK Signaling Axis.

Acute myeloid leukemia (AML) with nucleophosmin 1 (NPM1) mutations exhibits distinct biological and clinical features, accounting for approximately one-third of AML. Recently, the N 6-methyladenosine (m6A) RNA modification has emerged as a new epigenetic modification to contribute to tumorigenesis and development. However, there is limited knowledge on the role of m6A modifications in NPM1-mutated AML. In this study, the decreased m6A level was first detected and high expression of fat mass and obesity-associated protein (FTO) was responsible for the m6A suppression in NPM1-mutated AML. FTO upregulation was partially induced by NPM1 mutation type A (NPM1-mA) through impeding the proteasome pathway. Importantly, FTO promoted leukemic cell survival by facilitating cell cycle and inhibiting cell apoptosis. Mechanistic investigations demonstrated that FTO depended on its m6A RNA demethylase activity to activate PDGFRB/ERK signaling axis. Our findings indicate that FTO-mediated m6A demethylation plays an oncogenic role in NPM1-mutated AML and provide a new layer of epigenetic insight for future treatments of this distinctly leukemic entity.

Tumour-derived small extracellular vesicles suppress CD8+ T cell immune function by inhibiting SLC6A8-mediated creatine import in NPM1-mutated acute myeloid leukaemia.

Acute myeloid leukaemia (AML) carrying nucleophosmin (NPM1) mutations has been defined as a distinct entity of acute leukaemia. Despite remarkable improvements in diagnosis and treatment, the long-term outcomes for this entity remain unsatisfactory. Emerging evidence suggests that leukaemia, similar to other malignant diseases, employs various mechanisms to evade killing by immune cells. However, the mechanism of immune escape in NPM1-mutated AML remains unknown. In this study, both serum and leukemic cells from patients with NPM1-mutated AML impaired the immune function of CD8+ T cells in a co-culture system. Mechanistically, leukemic cells secreted miR-19a-3p into the tumour microenvironment (TME) via small extracellular vesicles (sEVs), which was controlled by the NPM1-mutated protein/CCCTC-binding factor (CTCF)/poly (A)-binding protein cytoplasmic 1 (PABPC1) signalling axis. sEV-related miR-19a-3p was internalized by CD8+ T cells and directly repressed the expression of solute-carrier family 6 member 8 (SLC6A8; a creatine-specific transporter) to inhibit creatine import. Decreased creatine levels can reduce ATP production and impair CD8+ T cell immune function, leading to immune escape by leukemic cells. In summary, leukemic cell-derived sEV-related miR-19a-3p confers immunosuppression to CD8+ T cells by targeting SLC6A8-mediated creatine import, indicating that sEV-related miR-19a-3p might be a promising therapeutic target for NPM1-mutated AML.

Mutant NPM1-regulated lncRNA HOTAIRM1 promotes leukemia cell autophagy and proliferation by targeting EGR1 and ULK3.

BACKGROUND: Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1), which displays a distinct long noncoding RNA (lncRNA) expression profile, has been defined as a unique subgroup in the new classification of myeloid neoplasms. However, the biological roles of key lncRNAs in the development of NPM1-mutated AML are currently unclear. Here, we aimed to investigate the functional and mechanistic roles of the lncRNA HOTAIRM1 in NPM1-mutated AML. METHODS: The expression of HOTAIRM1 was analyzed with a public database and further determined by qRT-PCR in NPM1-mutated AML samples and cell lines. The cause of upregulated HOTAIRM1 expression was investigated by luciferase reporter, chromatin immunoprecipitation and ubiquitination assays. The functional role of HOTAIRM1 in autophagy and proliferation was evaluated using western blot analysis, immunofluorescence staining, a Cell Counting Kit-8 (CCK-8) assay, a 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, flow cytometric analyses and animal studies. The action mechanism of HOTAIRM1 was explored through RNA fluorescence in situ hybridization, RNA pulldown and RNA immunoprecipitation assays. RESULTS: HOTAIRM1 was highly expressed in NPM1-mutated AML. High HOTAIRM1 expression was induced in part by mutant NPM1 via KLF5-dependent transcriptional regulation. Importantly, HOTAIRM1 promoted autophagy and proliferation both in vitro and in vivo. Mechanistic investigations demonstrated that nuclear HOTAIRM1 promoted EGR1 degradation by serving as a scaffold to facilitate MDM2-EGR1 complex formation, while cytoplasmic HOTAIRM1 acted as a sponge for miR-152-3p to increase ULK3 expression. CONCLUSIONS: Taken together, our findings identify two oncogenic regulatory axes in NPM1-mutated AML centered on HOTAIRM1: one involving EGR1 and MDM2 in the nucleus and the other involving the miR-152-3p/ULK3 axis in the cytoplasm. Our study indicates that HOTAIRM1 may be a promising therapeutic target for this distinct leukemia subtype.

[miR-148b-3p inhibits the proliferation and autophagy of acute myeloid leukemia cells by targeting ATG14].

Objective To investigate the effect of miR-148b-3p on the proliferation and autophagy of acute myeloid leukemia (AML) cells and its molecular mechanism. Methods Based on GEO and TCGA databases, the expression of miR-148b-3p in AML cells and its association with clinical prognosis of patients were analyzed with the bioinformatics software. The expression of miR-148b-3p in AML cells was detected by real-time quantitative PCR. The miR-148b-3p mimic and the miR-148b-3p inhibitor were transiently transfected into AML cell lines THP-1 and NB4 by liposome-mediated transfection, respectively. The proliferation of leukemia cells was evaluated by CCK-8 assay and 5-ethynyl-2'-deoxyuridine (EdU) labeling, and the protein levels of Bcl2, Bcl2-associated X protein (BAX), autophagy marker LC3, P62, and autophagy-related gene 14 (ATG14) were detected by Western blotting. The targeted binding of miR-148b-3p to ATG14 was measured by dual-luciferase reporter gene assay, and the effect of miR-148b-3p/ATG14 axis on the phenotype of AML cells was observed in the rescue experiments. Results A decreased expression of miR-148b-3p was found in leukemia blasts of AML patients, and the overall survival rate of AML patients with low expression of miR-148b-3p was significantly lower than that of the control group. Overexpression of miR-148b-3p inhibited THP-1 cells proliferation, promoted their apoptosis, downregulated the LC3II and ATG14 protein levels, and upregulated the P62 protein levels, while inhibiting the expression of miR-148b-3p in NB4 cells had the opposite effect. miR-148b-3p significantly reduced the luciferase activity of the wild-type ATG14 expression vector. The results of rescue experiments showed that overexpression of ATG14 reversed the inhibitory effect of miR-148b-3p upregulation on cell proliferation and autophagy, while inhibition of ATG14 expression weakened the promotive effect of miR-148b-3p downregulation on cell phenotype. Conclusion The miR-148b-3p inhibits the in vitro proliferation and autophagy of AML cells by targeting ATG14.

Hypoxia-stimulated ATM activation regulates autophagy-associated exosome release from cancer-associated fibroblasts to promote cancer cell invasion.

Cancer-associated fibroblasts (CAFs) as a predominant cell component in the tumour microenvironment (TME) play an essential role in tumour progression. Our earlier studies revealed oxidized ATM activation in breast CAFs, which is independent of DNA double-strand breaks (DSBs). Oxidized ATM has been found to serve as a redox sensor to maintain cellular redox homeostasis. However, whether and how oxidized ATM in breast CAFs regulates breast cancer progression remains poorly understood. In this study, we found that oxidized ATM phosphorylates BNIP3 to induce autophagosome accumulation and exosome release from hypoxic breast CAFs. Inhibition of oxidized ATM kinase by KU60019 (a small-molecule inhibitor of activated ATM) or shRNA-mediated knockdown of endogenous ATM or BNIP3 blocks autophagy and exosome release from hypoxic CAFs. We also show that oxidized ATM phosphorylates ATP6V1G1, a core proton pump in maintaining lysosomal acidification, leading to lysosomal dysfunction and autophagosome fusion with multi-vesicular bodies (MVB) but not lysosomes to facilitate exosome release. Furthermore, autophagy-associated GPR64 is enriched in hypoxic CAFs-derived exosomes, which stimulates the non-canonical NF-κB signalling to upregulate MMP9 and IL-8 in recipient breast cancer cells, enabling cancer cells to acquire enhanced invasive abilities. Collectively, these results provide novel insights into the role of stromal CAFs in promoting tumour progression and reveal a new function of oxidized ATM in regulating autophagy and exosome release.

GPER-Induced ERK Signaling Decreases Cell Viability of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive malignancy with a poor prognosis. Effective biomarkers and specific therapeutic targets for HCC are therefore urgently needed. G protein-coupled estrogen receptor (GPER) plays a crucial role in numerous cancer types; however, its functions in HCC require further exploration. In the present study, we found a remarkable difference in GPER staining between tumor tissue (100/141, 70.9%) and matched non-tumor tissue (27/30, 90.0%). Compared with the GPER-negative patients, the GPER-positive patients with HCC were closely associated with female sex, negative hepatitis B surface antigen, small tumor size, low serum alpha fetoprotein level, and longer overall survival. Treatment with GPER-specific agonist G1 led to the sustained and transient activation of the EGFR/ERK and EGFR/AKT signaling pathways, respectively, in the HCC cell lines HCCLM3 and SMMC-7721, which express high levels of GPER. Interestingly, G1-induced EGFR/ERK signaling, rather than EGFR/AKT signaling mediated by GPER, was involved in decreasing cell viability by blocking cell cycle progression, thereby promoting apoptosis and inhibiting cell growth. Clinical analysis indicated that simultaneous high expression of GPER and phosphorylated-ERK (p-ERK) predicted improved prognosis for HCC. Finally, the activation of GPER/ERK signaling remarkably suppressed tumor growth in an HCC xenograft model, and this result was consistent with the in vitro data. Our findings suggest that specific activation of the GPER/ERK axis may serve as a novel tumor-suppressive mechanism and that this axis could be a therapeutic target for HCC.

A Novel Long Non-Coding RNA lnc030 Maintains Breast Cancer Stem Cell Stemness by Stabilizing SQLE mRNA and Increasing Cholesterol Synthesis.

Cancer stem cells (CSCs) are considered the roots of cancer metastasis and recurrence (CSCs), due in part to their self-renewal and therapy resistance properties. However, the underlying mechanisms for the regulation of CSC stemness are poorly understood. Recently, increasing evidence shows that long non-coding RNAs (lncRNAs) are critical regulators for cancer cell function in various malignancies including breast cancer, but how lncRNAs regulate the function of breast cancer stem cells (BCSCs) remains to be determined. Herein, using lncRNA/mRNA microarray assays, a novel lncRNA (named lnc030) is identified, which is highly expressed in BCSCs in vitro and in vivo, as a pivotal regulator in maintaining BCSC stemness and promoting tumorigenesis. Mechanistically, lnc030 cooperates with poly(rC) binding protein 2(PCBP2) to stabilize squalene epoxidase (SQLE) mRNA, resulting in an increase of cholesterol synthesis. The increased cholesterol in turn actives PI3K/Akt signaling, which governs BCSC stemness. In summary, these findings demonstrate that a new, lnc030-based mechanism for regulating cholesterol synthesis and stemness properties of BCSCs. The lnc030-SQLE-cholesterol synthesis pathway may serve as an effective therapeutic target for BCSC elimination and breast cancer treatment.

NPM1 mutant maintains ULK1 protein stability via TRAF6-dependent ubiquitination to promote autophagic cell survival in leukemia.

Nucleophosmin (NPM1) mutations are the most frequent genetic alteration in acute myeloid leukemia (AML) and aberrant cytoplasm-dislocated NPM1 mutant is a distinct biological characterization of this disease. Our group previously reported that NPM1 mutant elevated autophagy activity and autophagy activation contributed to leukemic cell survival. However, the molecular mechanisms by which cytoplasmic NPM1 mutant involving in the autophagy pathway has not been fully elucidated. Here, we showed that Unc-51-like kinase 1 (ULK1) as a core autophagy protein was highly expressed in NPM1-mA positive OCI-AML3 cells and primary NPM1-mutated AML blasts. Meanwhile, we found that NPM1-mA could interact with ULK1 protein and positively regulated ULK1 protein levels. Mechanically, NPM1-mA promoted TRAF6-dependent K63 ubiquitination and further maintained ULK1 stability and kinase activity via miR-146a. In addition, ULK1 high expression-mediated autophagy activation and facilitated to leukemic cell proliferation. Finally, we demonstrated that restoring ULK1 expression, ULK1 inhibitor SBI-0206965 treatment and using shULK1 partially rescued the effect of NPM1-mA on autophagy and cell survival. In conclusion, our findings suggest that NPM1 mutant interacts with ULK1, and thus, maintains its protein stability, which is required for NPM1 mutant-mediated autophagic cell survival. These data extend our understanding of the functions of NPM1 mutant in the regulation of autophagy activation in NPM1-mutated AML.

Oxidized ATM promotes breast cancer stem cell enrichment through energy metabolism reprogram-mediated acetyl-CoA accumulation.

Cancer stem cell (CSC) is a challenge in the therapy of triple-negative breast cancer (TNBC). Intratumoral hypoxia is a common feature of solid tumor. Hypoxia may contribute to the maintenance of CSC, resulting in a poor efficacy of traditional treatment and recurrence of TNBC cases. However, the underlying molecular mechanism involved in hypoxia-induced CSC stemness maintenance remains unclear. Here, we report that hypoxia stimulated DNA double-strand breaks independent of ATM kinase activation (called oxidized ATM in this paper) play a crucial role in TNBC mammosphere formation and stemness maintenance by governing a specific energy metabolism reprogramming (EMR). Oxidized ATM up-regulates GLUT1, PKM2, and PDHa expressions to enhance the uptake of glucose and production of pyruvate rather than lactate products, which facilitates glycolytic flux to mitochondrial pyruvate and citrate, thus resulting in accumulation of cytoplasmic acetyl-CoA instead of the tricarboxylic acid (TCA) cycle by regulating ATP-citrate lyase (ACLY) activity. Our findings unravel a novel model of TNBC-CSC glucose metabolism and its functional role in maintenance of hypoxic TNBC-CSC stemness. This work may help us to develop new therapeutic strategies for TNBC treatment.

Drosha-independent miR-6778-5p strengthens gastric cancer stem cell stemness via regulation of cytosolic one-carbon folate metabolism.

Drosha-dependent canonical microRNAs (miRNAs) play a crucial role in the biological functions and development of cancer. However, the effects of Drosha-independent non-canonical miRNAs remain poorly understood. In our previous work, we found a set of aberrant miRNAs, including some upregulated miRNAs, called Drosha-independent noncanonical miRNAs, in Drosha-knockdown gastric cancer (GC) cells. Surprisingly, Drosha-silenced GC cells still retained strong malignant properties (e.g., proliferation ability and cancer stem cell (CSC) characteristics), indicating that aberrantly upregulated non-canonical miRNAs may play an important role in the maintenance of the malignant properties in GC cells that express low Drosha levels. Here, we report that miR-6778-5p, a noncanonical miRNA, acts as a crucial regulator for maintenance of CSC stemness in Drosha-silenced GC cells. MiR-6778-5p belongs to the 5'-tail mirtron type of non-canonical miRNAs and is transcript splice-derived from intron 5 of SHMT1 (coding cytoplasmic serine hydroxymethyltransferase). It positively regulates expression of its host gene, SHMT1, via targeting YWHAE in Drosha-knockdown GC cells. Similar to its family member SHMT2, SHMT1 plays a crucial role in folate-dependent serine/glycine inter-conversion in one-carbon metabolism. In Drosha wild type GC cells, SHMT2 mediates a mitochondrial-carbon metabolic pathway, which is a major pathway of one-carbon metabolism in normal cells and most cancer cells. However, in Drosha-silenced or Drosha low-expressing GC cells, miR-6778-5p positively regulates SHMT1, instead of SHMT2, thus mediating a compensatory activation of cytoplasmic carbon metabolism that plays an essential role in the maintenance of CSCs in gastric cancer (GCSCs). Drosha wild type GCSCs with SHMT2 are sensitive to 5-fluorouracil; however, Drosha low-expressing GCSCs with SHMT1 are 5-FU-resistant. The loss of miR-6778-5p or SHMT1 notably mitigates GCSC sphere formation and increases sensitivity to 5-fluorouracil in Drosha-knockdown gastric cancer cells. Thus, our study reveals a novel function of Drosha-independent noncanonical miRNAs in maintaining the stemness of GCSCs.

GPER mediates decreased chemosensitivity via regulation of ABCG2 expression and localization in tamoxifen-resistant breast cancer cells.

Rescue chemotherapy is usually the preferred treatment for patients with advanced estrogen receptor-positive (ER+) breast cancer with endocrinotherapy resistance. However, these patients often simultaneously show a poor response to cytotoxic drugs, and thus the detailed mechanism of this resistance needs to be further investigated. Our previous research indicated that the G-protein-coupled estrogen receptor (GPER) is a novel mediator of the development of multidrug resistance, including resistance to both endocrinotherapy and chemotherapy, and ATP binding cassette subfamily G member 2 (ABCG2) has been identified as an engine that confers cancer cells with chemoresistance by expelling xenobiotics and chemotherapeutics. Here, we are the first to show that the expression levels of GPER and ABCG2 are markedly increased in tamoxifen-resistant ER + metastases compared to the corresponding primary tumors. A plasma membrane expression pattern of GPER and ABCG2 was observed in patients with metastases. Furthermore, both ER modulator tamoxifen, GPER-specific agonist G1 and pure ER antagonist ICI 182,780 significantly enhanced ABCG2 expression in tamoxifen-resistant breast cancer cells (MCF-7R) but not in tamoxifen-sensitive cells (MCF-7). The activated downstream GPER/EGFR/ERK and GPER/EGFR/AKT signaling pathways were responsible for regulating the expression and cell membrane localization of ABCG2, respectively, in MCF-7R cells. Interestingly, the above phenomenon could be alleviated by inhibitors of both the indicated signaling pathways and by knockdown of GPER in MCF-7R cells. More importantly, the tamoxifen-induced GPER/ABCG2 signaling axis was shown to play a pivotal role in the development of chemotherapy (doxorubicin) resistance both in vitro and in vivo. The clinical data further revealed that tamoxifen-resistant patients with high GPER/ABCG2 signaling activation had poor progression-free survival (PFS) when given rescue anthracycline chemotherapy. Therefore, our data provide novel insights into GPER-mediated chemoresistance and provide a rationale for the GPER/ABCG2 signaling axis being a promising target for reversing chemoresistance in patients with advanced ER + tamoxifen-resistant breast cancer.

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

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 17ß-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.