Research ArticleExperimental Studies
Open Access

Suppression of G Protein-coupled Estrogen Receptor 1 (GPER1) Enhances the Anti-invasive Efficacy of Selective ERβ Agonists

VIVIEN SCHMITZ, GERD BAUERSCHMITZ, JULIA GALLWAS and CARSTEN GRÜNDKER
Anticancer Research November 2022, 42 (11) 5187-5194; DOI: https://doi.org/10.21873/anticanres.16025

Abstract

Background/Aim: G protein-coupled estrogen receptor 1 (GPER1) is often over-expressed in triple negative breast cancer (TNBC). GPER1 is responsible for many of the non-genomic, membrane-initiated effects of estrogens. Therefore, we have analyzed the effects of GPER1 knockdown using specific siRNA. Materials and Methods: Transient GPER1 silencing was conducted using RNA interference and confirmed by RT-PCR and western blot. Viability of human breast cancer cell lines MDA-MB 231 and HCC 1806 was tested using AlamarBlue assay. Cell invasion was analyzed by assessment of cell migration rate through an artificial basement membrane in a modified Boyden chamber. Results: Viability of both cell lines was slightly decreased after suppression of GPER1 expression. Knockdown of GPER1 resulted in a significantly reduced invasion of the TNBC cells. The anti-invasive effect of selective ERβ agonists was significantly stronger after knockdown of GPER1 expression. In addition, the efficacy of tamoxifen treatment was significantly increased after suppression of GPER1 expression. Conclusion: Suppression of GPER1 reduced the metastatic behavior of TNBC cells, improved the anti-invasive efficacy of selective ERβ agonists and sensitized cells to 4OH-tamoxifen.

Key Words:

In malignant breast cancer cells, estrogen receptor beta (ERb) has anti-proliferative and anti-invasive effects. Accordingly, a significantly better survival of patients with ERb positive triple-negative breast cancer (TNBC) compared to ERb negative TNBC can be achieved. ERb not only serves as a prognostic marker, but represents also a possible target for the therapy of TNBC. Previous studies have successfully treated TNBC cells with specific ER agonists (1).

For example, treatment of cell lines with the specific ERb agonists Liquiritigenin and ERB-041, led to a significant decrease in invasion (1). A further study has shown a decrease in tumor cell proliferation after treatment (2). Liquiritigenin is a herbal compound extracted from a Chinese liquorice plant, Glycyrrhiza uralensis. It has anti-inflammatory, antimicrobial, antioxidant, immunomodulatory, antiproliferative, and anti-invasive properties. In breast cancer tumors, it prevents angiogenesis and proliferation via the vascular endothelial growth factor receptor (VEGFR) signaling pathway by directly inhibiting VEGF via hypoxia inducible factor-1alpha (HIF1 alpha). Liquiritigenin is seen as a promising therapeutic of TNBC due to the further inhibiting effects in estradiol-mediated signaling pathways (3). Erb-041 is a synthetically produced, non-steroidal compound that binds highly specifically to the ERβ receptor. Compared to liquiritigenin, ERB-041 is also considered to have an anti-invasive and anti-inflammatory effect, but no anti-proliferative property could be determined (4).

G protein-coupled estrogen receptor 1 (GPER1) is a transmembrane receptor that is stimulated by estradiol and, coupled with G-protein, activates an intracellular signal cascade. It mediates cell responses that do not interfere with the cell’s gene expression. The GPER1-mediated cell responses are much faster, within seconds to minutes. When the membrane-bound estrogen receptor is activated, many signal cascades run simultaneously within the cell, which communicate with one another and also activate themselves (5). The adenylate cyclase is activated, which provides cAMP with the help of ATP. High intracellular cAMP concentrations are responsible for a genome-independent cell response, but can also influence gene expression by activating protein kinase A. GPER1 also recruits high levels of intracellular calcium via phospholipase C. Calcium assumes multiple functions within the cell, activating enzymes, hormone secretion, and muscle contraction. It in turn activates the mitogen-activated protein kinase (MAP kinase) ERK, which induces cell transcription. This is also activated by epidermal growth factor (EGF). EGF is a G-protein coupled via the Scr kinase activated by matrix metalloproteases (MMP). In addition to the activation of ERK, phosphoinisitol-3-kinase (PI3) is also activated. After phosphorylation of Akt kinase, the transcription factor forkhead box receptor 3 (FOXO3) is recruited. FOXO3 is a tumor suppressor gene that protects the cell from degeneration. After phosphorylation, FOXO3 is inhibited. GPER1 agonists can thus cause cell degeneration. Studies have shown that TNBC patients with high expression of GPER1 have a reduced survival time compared to TNBC patients with low expression of GPER1. Furthermore, after GPER1 knockdown in TNBC cell lines, estradiol-dependent cell growth, Src kinase, and EGFR were significantly inhibited. GPER1 antagonists are considered a possible therapeutic option for TNBC. Estriol (E3), which completely inhibits GPER1 (6-8), acts as a possible GPER1 antagonist in TNBC.

The selective estrogen receptor modulator (SERM) tamoxifen is an antagonist for ERa in mammary gland tissue, but binds to GPER1 receptor with high affinity and acts there as an agonist and plays an important role in the development of tamoxifen resistance in breast cancer. The intracellular signal cascades in breast cancer cells are activated, and proliferation and invasion are promoted (5). Studies have shown that the majority of tamoxifen-resistant breast cancers (BC-TR) also express GPER1 (6). Studies have already shown that specific ERb agonists have an anti-invasive effect on TNBC cells (9). Furthermore, it was found that a lower expression of GPER1 is associated with a better prognosis for TNBC (10).

Knowledge of the relationships between GPER1, ERα, ERβ and the effectiveness of tamoxifen or resistance to tamoxifen is still partially incomplete. The aim of the present work was therefore to investigate to what extent suppression of GPER1 affects viability and invasiveness of TNBC cells. In addition, it was of interest to determine whether suppression of GPER1 affects the efficacy of selective ERβ agonists and the SERM tamoxifen.

Materials and Methods

Cell culture. The human breast cancer cell lines MDA-MB 231 and HCC 1806 were obtained from the American Type Cell Collection (ATCC; Manassas, VA, USA) and cultured in minimum essential medium (MEM; Biowest, Nuaillé, France) supplemented with 10% fetal bovine serum (FBS; Biochrom, Berlin, Germany), 1% Penicillin/Streptomycin (P/S; Gibco, Carlsbad, CA, USA), 0.1% Transferrin (Sigma, St. Louis, MO, USA), and 26 IU Insulin (Sanofi, Frankfurt, Germany). Human osteosarcoma cell line MG-63 was purchased from ATCC and cultured with Dulbecco’s modified eagle medium (DMEM; Gibco) supplemented with 10% FBS (Biochrom) and 1% Penicillin/Streptomycin (Gibco). To retain the identity of cell lines, purchased cells were expanded and aliquots were frozen in liquid nitrogen. A new frozen stock was used every half year and mycoplasma testing of cultured cell lines was performed routinely using PCR Mycoplasma Test Kit I/C (PromoCell GmbH, Heidelberg, Germany). All cells were cultured in a humidified atmosphere with 5% CO2 at 37°C.

Small interfering RNA transfection. The breast cancer cell lines MDA-MB 231 (1×106 cells/ml) and HCC1806 (5×105 cells/ml) were seeded in 2 ml of MEM with 10% FBS (−P/S) in a 25 cm2 cell culture flask. Cells were transiently transfected with small interfering RNA (siRNA) specific to GPER1 (sc-60743; Santa Cruz Biotechnology, Dallas, TX, USA) in OPTI-MEM I medium (Gibco) with siRNA transfection reagent (sc-29528; Santa Cruz Biotechnology). A non-targeting control was used as a control (sc-37007; Santa Cruz Biotechnology). In order to evaluate the transfection efficiency, a fluorescein-labeled siRNA control (sc-36869; Santa Cruz Biotechnology) was used. After an incubation period of 6 h, MEM supplemented with 20% FBS and 20% penicillin/streptomycin was added.

Co-culture transwell vertical invasion assay. Using the co-culture transwell vertical invasion assay (11), as described earlier (12), 1×104 breast cancer cells were seeded in DMEM w/o phenol red, supplemented with 10% charcoal stripped fetal calf serum (cs-FCS) into a cell cultural insert (upper well) with a polycarbonate membrane (8 μm pore diameter, Merck Millipore, Cork, Ireland) coated with 30 μl of mouse Engelbreth-Holm-Swarm (EHS) sarcoma-derived basement membrane matrix gel called, Matrigel® (2.4 mg/ml, BD Bioscience, Bedford, MA, USA) solution (1:2 in serum-free DMEM) or gelatin (1 mg/ml in PBS, Sigma). Osteosarcoma cells were seeded (2.5×104) in DMEM supplemented with or without 10% cs-FCS into the lower well (24-well-plate). After 24 h, cells were co-cultured for 96 h. Invaded cells on the lower side of the inserts were stained with hematoxylin, and the number of cells in four randomly selected fields of each insert was counted.

Viability assay. The breast cancer cells were seeded in 96 well plates (1.25×103) in DMEM w/o phenol-red supplemented with 10% cs-FBS, and relative AlamarBlue reduction (BioRad, Hercules, CA, USA) was assessed at 120 h. Thereafter, relative AlamarBlue reduction was measured by absorbance readings at 540 and 630 nm using Synergy (BioTek Instruments). Relative AlamarBlue Reduction was calculated as indicated by the manufacturer.

PCR analysis. Total RNA was extracted using a RNeasy mini kit (Qiagen, Hilden, Germany), and 2 μg was reverse transcribed with a high-capacity cDNA reverse transcription kit (Qiagen). RT-PCR was performed as previously described (6). PCR-products were separated in a 2% agarose gel (Type IV, special high EEO; Sigma). Gels were stained in ethidium bromide (2 lg/ml) for 30 min and photographed on a transilluminator using a CDS camera (Biometra, Göttingen, Germany). The band intensities of the RT-PCR products were evaluated by the Digital science 1D-software (Kodak, Rochester, NY, USA). Values of the RT-PCR products were normalized to the ribosomal protein L7.

Western blot analysis. In western blot analysis, cells were lysed in cell lytic M buffer (Sigma) supplemented with 0.1% phosphatase-inhibitor (Sigma) and 0.1% protease-inhibitor (Sigma). Isolated proteins (40 μg) were fractioned using 12% SDS gel and electrotransferred to a polyvinylidene difluoride membrane (Merck Millipore, Cork, Ireland). Primary antibodies against GPER1 1:1,000 (PA5-28647, Thermo Fisher Scientific, Waltham, MA, USA), and GAPDH 1:2,000 (5174S, Cell Signaling) were used. The membrane was washed and incubated in horseradish peroxidase-conjugated secondary antibody (GE Healthcare, Buckinghamshire, UK). Antibody-bond protein bands were assayed using a chemiluminescent luminol enhancer solution (Cyanagen, Bologna, Italy).

Statistical analysis. All experiments were performed on at least three biological and technical replicates. Data were analyzed by GraphPad Prism Software version 8.41 (GraphPad Software Inc., La Jolla, CA/USA) using unpaired, two-tailed, parametric t-tests comparing two groups (treatment to respective control) by assuming both populations had the same standard derivation or with an ANOVA one-way analysis when more than two groups were compared. F-values were recorded, and a Dunnett‘s or a Tukey‘s multiple comparison test with no matching or pairing between groups was calculated. p<0.05 was considered statistically significant.

Results

In order to examine whether GPER1 is a suitable target for TNBC therapy, we analyzed the effects of suppression of GPER1 expression in TNBC cell viability, invasion, estrogen receptor expression, and anti-invasive efficacy of selective ERβ agonists. We also checked whether the TNBC cells became sensitive to the tamoxifen after down-regulating GPER1.

Suppression of GPER1 expression. First, we tested whether GPER1 expression can be effectively reduced by siRNA. Suppression of GPER1 mRNA expression was detectable as early as 24 h after siRNA transfection and became significant in MDA-MB 231 cells after 96 h (0.43±0.17, p<0.05 vs. control, Figure 1A). GPER1 mRNA suppression was significant in HCC1806 cells 24 h after transfection (0.60±0.08, p<0.05 vs. control, Figure 1C). Suppression of GPER1 mRNA in HCC1806 cells remained reduced up to 168 h but was no longer significant. Suppression of GPER1 protein expression was tested at 96 h and was significant in both cell lines (MDA-MB-231: 49.33±4.81%, p< 0.05 vs. control, Figure 1B; HCC1806: 17.00±4.58%, p<0.0.0001 vs. control, Figure 1D).

Figure 1.
Figure 1.

Suppression of GPER1 mRNA (A, C) and protein (B, D) expression by siRNA interference in triple-negative breast cancer cell lines MDA-MB-231 (A, B) and HCC1806 (C, D). Columns represent means±SEM of data obtained from three independent experiments in three different passages of the cell lines (unpaired t-test).

Effects of GPER1 suppression on viability and cell invasion. The next step was to verify whether suppression of GPER1 had an effect on the efficacy of selective ERβ agonists. The selective ERβ agonists ERB-041 and liquiritigenin only marginally reduced the viability of both cell lines (Figure 2A and B). Suppression of GPER1 had no effect on this (Figure 2A and B). Suppression of GPER1 per se also resulted in a very small reduction in viability. Furthermore, invasion of MDA-MB-231 cells was not affected by treatment with the selective ERβ agonists (Figure 2C, while treatment of HCC1806 cells resulted in a slight decrease in invasion (ERB-041: 81.94±9.19%; liquiritigenin: 80.41±8.04%; Figure 2D), an effect that was significantly enhanced by suppression of GPER1 expression. After GPER1 knockdown, a particularly strong anti-invasive effect was detectable by treatment of HCC1806 cells with the selective ERβ agonists (ERB-041: 39.10±2.27%, p<0.001 vs. siRNA control, p<0.001 vs. GPER1 suppression alone, p<0.01 vs. ERB-041 alone; liquiritigenin: 42.50±4.06 %, p<0.01 vs. siRNA control, p<0.05 vs. GPER1 suppression alone, p<0.05 vs. liquiritigenin alone; Figure 2D). Even invasion of MDA-MB-231 cells was significantly reduced by treatment with the selective ERβ agonists after GPER1 suppression (ERB-041: 42.50±3.63%, p<0.001 vs. siRNA control, p<0.05 vs. GPER1 suppression alone, p<0.01 vs. ERB-041 alone; liquiritigenin: 32.79±1.89%, p<0.0001 vs. siRNA control, p<0.01 vs. GPER1 suppression alone, p<0.0001 vs. liquiritigenin alone; Figure 2C).

Figure 2.
Figure 2.

Effects of GPER1 suppression and selective ERβ agonist treatment on viability (A, B) and cell invasion (C, D) of triple-negative breast cancer cell lines MDA-MB-231 (A, C) and HCC1806 (B, D). Columns represent means±SEM of data obtained from three independent experiments in three different passages of the cell lines (ANOVA followed by Tukey’s multiple comparisons test).

Effects of GPER1 suppression on 4OH-tamoxifen efficacy. Since GPER1 seems to play a role in tamoxifen-resistance, it was of interest to examine whether SERMs, such as tamoxifen might be effective in the MDA-MB-231 and HCC1806 TNBC cells after suppression of GPER1 expression. While tamoxifen had no effect on the viability of both cell lines treated with control siRNA (Figure 3A and C), tamoxifen treatment resulted in a significant reduction in viability of both cell lines after suppression of GPER1 expression (MDA-MB-231: 68.67±9.09%, p<0.01 vs. GPER1 suppression; Figure 3B; HCC1806: 54.17±7.41%, p<0.001 vs. GPER1 suppression alone; Figure 3D).

Figure 3.
Figure 3.

Effects of GPER1 suppression on 4OH-tamoxifen efficacy on viability of triple-negative breast cancer cell lines MDA-MB-231 (A, B) and HCC1806 (C, D). Columns represent means±SEM of data obtained from three independent experiments in three different passages of the cell lines (unpaired t-test).

Discussion

In TNBC cells, expression of ERβ as well as membrane-bound GPER1 is increased. In breast cancer tamoxifen-resistant (BC-TR) cells GPER1 expression is also up-regulated (13, 14). Continuous treatment of breast cancer cells with tamoxifen increases GPER1 expression (15). In addition, expression of GPER1 in metastases was reported to be higher than that in corresponding primary tumors (16, 17). In this context, a correlation was found between GPER expression and worse clinical-pathological features of BC, although controversial data were also reported (18).

Treatment with specific ERβ agonists leads to a significantly reduced tendency to metastasize (1). Furthermore, after estrogen treatment, the cancer cells show a rapid cellular response, which is associated with proliferation and invasion. This cannot be explained by the much slower cellular response to estrogen treatment normally permitted by nuclear estrogen receptors. The rapid response to estrogen treatment occurs due to activation of the transmembrane receptor GPER1, which activates multiple signaling cascades and transcription factors independent of nuclear estrogen receptors (7).

We examined the effect of suppressing GPER1 on the viability and invasiveness of TNBC cells and how this affects the efficacy of therapy with selective ERβ agonists or the SERM tamoxifen? Since cell motion is not identical in two-dimensional (2D) and 3D models, we analyzed invasion using a vertical transwell invasion assay with Matrigel. This 3D mimicking tumor microenvironment model at least partially bridges the gap between 2D and the matrix structure surrounding cancer cells in vivo, as well as the stiffness of the microenvironment as an important rheological parameter modulating cell migration, invasion, and cell-cell adhesion (11).

After GPER1 knockdown, ERβ expression in both TNBC cell lines was briefly reduced but then returned to control levels (not shown). TNBC is an imprecise subtype of breast cancer of heterogeneous nature, which shows negative immunohistochemical staining for ERα, PR, and Her-2. The cell lines MDA-MB-231 and HCC1806 used in this study also show negative immunohistochemical staining for ERα, PR, and Her-2 (19-21). Depending on the definition, ERα expression, assayed using immunocytochemistry, in TNBC cells ranges between <1% and <10% (22). A natural heterogeneity in receptor expression is ascribed to TNBC cells. According to this, TNBC cells differ in their expression of receptors despite having the same molecular subtype (23).

While suppression of GPER1 and treatment with selective ERβ agonists showed no effect on TNBC cell viability, cell invasion was significantly affected. Both cell lines showed a decreased propensity to invade after knockdown of GPER1 compared to control, with the more invasive HCC 1806 cells showing a higher and significant decrease in invasion after GPER1 suppression.

The selective ERβ agonists had an anti-invasive effect on the TNBC cell line HCC1806 as has also been shown by Hinsche et al. (1). In contrast, no anti-invasive effect was detected on the TNBC cell line MDA-MB-231. Suppression of GPER1 expression enhanced the anti-invasive effect of selective ERβ agonists. In conjunction with GPER1 suppression, ERβ agonists also exerted anti-invasive effects on the MDA-MB-231 cell line, which was significantly stronger than that of GPER1 knockdown alone. Thus, suppression of GPER1 enhances the efficacy of selective ERβ agonists without permanently altering ERβ expression.

Treatment with ERb agonists, as well as suppression of GPER1, have a synergistic, anti-invasive effect on TNBC cells. Signaling via GPER1 is inhibited by its silencing. Accordingly, a pro-invasive and proliferative cell response was not observed. ERb agonists specifically activate ERb. This promotes cell differentiation, apoptosis, cell cycle arrest and autophagy of degenerated cells and inhibits transcription and cell growth of malignant breast cancer cells (24, 25). Other studies have already highlighted that EGFR plays a key role in the interaction of estradiol with various estrogen receptors and their activation. EGFR is activated directly after activation of GPER1 by estradiol. This accelerates cell replication via multiple intercommunicating signaling pathways. EGF gene expression is induced via activation of the nuclear estrogen receptors, whereas the rapid cellular responses regarding cell proliferation and gene expression are induced via activation of the membrane-bound GPER1. If GPER1 is suppressed, direct activation of EGFR is prevented, and a limited E2-dependent cell response is observed.

ERa positive tumors show increased expression of EGFR as they receive their growth stimulus specifically from estrogens (26). Estradiol can directly activate ERa and thus promotes proliferation of ERa positive tumors (27). Increased EGFR expression is associated with a worse prognosis of such tumors. Furthermore, in TNBC cells, the ERa antagonist tamoxifen has been shown to directly activate GPER1 (28). Accordingly, tamoxifen treatment activates EGFR resulting in an increase in the proliferation of malignant cells. Long-term treatment of ERa positive breast cancer cells with tamoxifen increases expression of GPER1 and cell proliferation (15). Only about 70% of ERa positive tumors respond to tamoxifen treatment (29). This phenomenon can be explained by the agonistic effect at GPER1 and the resulting EGFR activation. Consequently, cell growth does not decline with increased GPER1/EGFR expression. Furthermore, it has been shown that the non-response to ERa antagonists is complex. Apart from a genetic heterogeneity of ER expression and resistance development in breast carcinomas, the variable levels of different transcription factors, the different affinity of the receptors to their ligands and the expression of receptor specific domains play an essential role (26). Cheng et al. (27) describe that tamoxifen down-regulates ERb by forming a tamoxifen-ERb complex. In the context of tamoxifen resistance, in turn, an increased expression of ERb could be demonstrated.

In this study, we demonstrated that after suppression of GPER1, TNBC cells become sensitized to tamoxifen. This means that suppression of GPER1 could be an option for the treatment of TNBCs but tamoxifen-resistant breast carcinomas in general. This needs to be tested in breast carcinomas that have developed resistance to tamoxifen. Liu et al. have shown that activation of GPER1 in breast cancer-associated fibroblasts induces accumulation of high-mobility-group-protein B1 (HMGB1) and, thus, enhances the resistance to tamoxifen by a paracrine effect (30). They propose to target GPER1 or its downstream signaling to attenuate the resistance of ERα-positive breast tumors to tamoxifen.

Owing to the inadequacy of therapeutic options in TNBC, interruption of the GPER1 signaling pathway may represent an important target for the reduction of tumor growth and invasion. Activation of GPER1 in TNBC cells leads to increased expression of GPER1 via a positive feedback loop, further accelerating the proliferation of TNBC cells. EGF can directly influence GPER1 gene expression via induction of ERK phosphorylation, c-fos expression and recruitment of transcription factor activator protein (AP-1). TNBC with GPER1 over-expression is characterized by poorer survival, and more aggressive tumor progression (26). In vivo, estriol (E3) exerts an inhibitory effect on GPER1. It can be hypothesized that GPER1 is specifically activated by estradiol, as in vitro experiments showed that GPER1 is inhibited after addition of E3 and cell proliferation of malignant TNBC cells is reduced (7). Furthermore, Chen et al. (31) observed that baicalein can bind ER and GPER1 and inhibit E2-dependent signaling responses. E2-dependent migration, adhesion and invasion of triple negative and ER positive breast cancer cells are inhibited. Therefore, baicalein represents a therapeutic option in the treatment of malignant breast carcinoma (31).

Currently, breast carcinomas are often examined histologically only in terms of ERa expression. Many studies have already highlighted that ERb expression has an impact on tumor progression. Depending on the expression of the respective subtype of ERb, different signaling pathways are regulated. By using specific antibodies against the ERb isoforms, clinical pathological parameters could be established, showing a correlation with corresponding carcinoma types (29). Therefore, ERb expression in breast tumors is considered an important prognostic factor and should be included in the standardized histopathological examination. Furthermore, the use of specific ERb agonists should be investigated as a therapeutic option in TNBC.

Previous research showed that TNBCs express ERβ and GPER1. The primary aim of this study was to highlight the extent to which suppression of GPER1 affects the anti-invasive effectiveness of specific ERβ agonists. It was shown that suppression of GPER1 leads to a decrease in the invasion of TNBC and that treatment of GPER1 knockdown cells with specific ERβ agonists causes a further reduction in invasion. The knockdown of GPER1 and the activation of ERβ by specific ERβ agonists had a synergistic and significantly stronger effect.

Furthermore, after suppression of GPER1, the TNBC cells were sensitized to treatment with tamoxifen. Additional studies must be carried out with ERα-positive breast cancer cells and their subclones with acquired resistance against tamoxifen. This means that suppression of GPER1 could represent a treatment option for TNBCs and tamoxifen-resistant breast cancers.

Regarding targeted therapy for TNBC, GPER1 and ERβ represent promising therapeutic targets. Refined histopathological examination of tumor tissues should precisely specify the estrogen receptors to allow for the best possible therapeutic approach. GPER1 may become a potential target to overcome tamoxifen resistance in the future.

Acknowledgements

The Authors thank Sonja Blume for the excellent technical assistance.

Footnotes

  • Authors’ Contributions

    Conceptualization, Carsten Gründker; Investigation, Vivien Schmitz; Project administration, Carsten Gründker; Writing original draft, Carsten Gründker; Review & editing, Gerd Bauerschmitz and Julia Gallwas.

  • Conflicts of Interest

    The Authors declare no conflicts of interest in relation to this study.

  • Received August 31, 2022.
  • Revision received September 18, 2022.
  • Accepted September 19, 2022.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Suppression of G Protein-coupled Estrogen Receptor 1 (GPER1) Enhances the Anti-invasive Efficacy of Selective ERβ Agonists
VIVIEN SCHMITZ, GERD BAUERSCHMITZ, JULIA GALLWAS, CARSTEN GRÜNDKER
Anticancer Research Nov 2022, 42 (11) 5187-5194; DOI: 10.21873/anticanres.16025
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