Research ArticleArticles
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G Protein-coupled Estrogen Receptor 1 (GPER1) Regulates Expression of SERPINE1/PAI-1 and Inhibits Tumorigenic Potential of Cervical Squamous Cell Carcinoma Cells In Vitro

LINEA RÖRIG, SOPHIA RUCKRIEGL, JULIA GALLWAS and CARSTEN GRÜNDKER
Cancer Genomics & Proteomics January 2025, 22 (1) 13-23; DOI: https://doi.org/10.21873/cgp.20482

Abstract

Background/Aim: G protein-coupled estrogen receptor 1 (GPER1) appears to play a tumor-suppressive role in cervical squamous cell carcinoma (CSCC)GPER1 suppression leads to significantly increased expression of serpin family E member 1 (SERPINE1)/protein plasminogen activator inhibitor type 1 (PAI-1). The question arises, what role does SERPINE1/PAI-1 play in GPER1-dependent tumorigenic potential of CSCC. Materials and Methods: SiHa and C33A CSCC cells were treated with GPER1 agonist G1 or antagonist G36. SERPINE1/PAI-1 expression was suppressed by RNAi and success was confirmed by RT-qPCR. Protein expression of PAI-1 was quantified by Western blot. Viability was analyzed using resazurin assay, while migration was investigated using gap closure. Colony and tumor sphere formation were used to test clonogenicity. Results: After G1 treatment, viability of SiHa and C33A cells remained unchanged. Cell migration was dose-dependently reduced. SiHa and C33A cells formed significantly fewer and smaller colonies as well as spheroids. Furthermore, treatment with G1 led to decreased expression of SERPINE1/PAI-1, while blockade of GPER1 with G36 resulted in significantly increased SERPINE1/PAI-1 expression. After suppression of SERPINE1/PAI-1 in SiHa cells using RNAi, cell viability remained unaffected; however, significantly smaller colonies were formed, and fewer and smaller spheroids were developed. Cell migration remained unaffected. Conclusion: Activation of GPER1 reduces clonogenicity and migration of CSCC cells and suppresses expression of SERPINE1/PAI-1. Suppression of SERPINE1/PAI-1 in CSCC cells reduces tumorigenic potential. GPER1 may be a suitable target for suppression of SERPINE1/PAI-1 in CSCC. However, SERPINE1/PAI-1 does not appear to be the decisive factor for GPER1-regulated cell migration.

Key Words:

Cervical carcinoma (CC) is the fourth most common cancer in women worldwide affecting mostly social minorities with lower socioeconomic status and less medical care and access to vaccinations (1). High-risk human papillomaviruses (HPV) are the cause of more than 99% of CCs (2). Most CCs are represented by the squamous cell type which accounts for around 80%, while adenocarcinomas account for 5-20% of carcinomas. Rarely, sarcomas or neuroendocrine tumors may occur (3).

The investigation of estrogen receptors is becoming increasingly important in current tumor research. The G protein-coupled estrogen receptor 1 (GPER1, formerly GPR30) is a seven-transmembrane-domain receptor recognized as a rapid mediator of cellular estrogenic action (4). GPER1 is found throughout the body, including the heart, brain, pancreas, kidneys, skeletal muscles as well as in blood vessels and in reproductive organs (5). It is mainly found in the endoplasmic reticulum (6). In CC, GPER1 was detected in the cytoplasm and in the nucleus of epithelial cells (7). GPER1 has been shown to act in many signaling pathways associated with cancer, including the mitogen-activated protein kinase, phosphoinositide 3-kinase, and the Hedgehog pathway. All effects are dependent on tissue type and environment (8). Ligands of GPER1 include the ubiquitous estrogens of the body, estrone and estradiol, as well as some drugs, such as raloxifene, tamoxifen and bisphenol A (6). Several other selective GPER1 ligands are now available for research (5).

We recently were able to show that GPER1 plays a tumor-suppressive role in CC. After suppression of GPER1 by siRNA, significant tumor-promoting effects were shown regarding colony formation, tumor sphere formation and invasiveness. Immunofluorescence staining revealed a strong expression of GPER1 in peripheral regions and in the sprouts of tumor spheres. After analyzing various prometastatic factors, it was shown that GPER1 knockdown leads to significantly increased expression of the oncogene serpin family E member 1 (SERPINE1) and the corresponding protein plasminogen activator inhibitor-1 (PAI-1) (9).

SERPINE1 gene and its protein product PAI-1, which were discovered in the 1970s, belong to the serin proteinase inhibitors and are primarily known to regulate fibrinolysis (10). PAI-1 is ubiquitous in the body and is found in many cell types, such as megakaryocytes, platelets, hepatocytes, adipocytes, smooth muscle cells and endothelial cells. In the bloodstream, PAI-1 is found in plasma and in platelet α-granules (10). The inhibition of PAI-1 activity also seems to attenuate lung fibrosis (11).

Furthermore, SERPINE1 is overexpressed in nine types of cancer (12). The malignant effect of SERPINE1 in gastric carcinoma has been demonstrated in various experiments (13). In gastric carcinoma, SERPINE1 has already been tested as a component of a risk score. Evidence suggests that the expression of SERPINE1 and other metabolism-associated genes is associated with a poor prognosis (14).

The findings about the tumor suppressive effects of GPER1 in CC and the up-regulation of SERPINE1/PAI-1 under GPER1 knockdown were the starting point of this research work (9). In this study, we investigated whether SERPINE1/PAI-1 is involved in the GPER1-mediated regulation of cervical squamous cell carcinoma (CSCC) progression, the most common type of CC. A connection between GPER1 and SERPINE1/PAI-1 could be interesting for future therapies that either target GPER1 or SERPINE1/PAI-1.

Materials and Methods

Cell culture. The human cervical carcinoma cell lines SiHa (HPV16+) and C33-A (HPV−) were obtained from the American Type Cell Collection (ATCC; Manassas, VA, USA) and cultured in minimum essential medium (MEM; L0416-500, Biowest, Nuaillé, France) supplemented with 10% fetal bovine serum (FBS; S181B-500, Biochrom, Berlin, Germany) and 1% penicillin/streptomycin (P/S; L0022-100, Biowest). To retain the identity of the 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 the polymerase chain reaction (PCR) Mycoplasma Test Kit I/C (D101-02, Vazyme, Düsseldorf, Germany). All cells were cultured in a humidified atmosphere with 5% CO2 at 37°C.

Treatments. The cells were treated with GPER1 agonist G1 or antagonist G36 (Biomol, Hamburg, Germany) in final concentrations of 1 μM, 2.5 μM, and 5 μM. The GPER1 analogs were dissolved in ethanol (AppliChem, Renningen, Germany). Each control was treated with 0.03 v/v% ethanol.

Cell viability. For the Resazurin assay, 10,000 cells per ml were seeded in 96-well plates (Corning Life Sciences, Amsterdam, the Netherlands) in Dulbecco’s Minimum Essential Medium w/o phenol-red (DMEM; Thermo Fisher Scientific, Waltham, MA, USA). Treatment with G1 or G36 was carried out 24 h later. After incubation for 72 h, 20 μl Resazurin (Thermo Fisher Scientific) was added to every well. Ten hours later, the relative reduction to Resorufin was measured at 570 nm and 630 nm using a multidetection microplate reader (BioTek Instruments, Bad Friedrichshall, Germany) and analyzed using GEN5 1.08 software (BioTek Instruments).

Gap closure. One hundred thousand cells per well (1,000,000 cells/ml) were seeded in a 24-well plate (Corning, Kennebunk, ME, USA) separated by an insert. After incubation for 24 h, the cells were washed with Dulbecco’s Phosphate Buffered Saline (DPBS; Pan Biotech) and treated with G1 or G36. Pictures were taken several times until the gap was closed using uEye Cockpit 2.0 (IDS Imaging Development Systems, Obersulm, Germany). Gap closure was analyzed by using ImageJ 1.52a (Wayne Rasband, National Institute of Health, Bethesda, MD, USA).

Colony formation. One thousand cells per well were seeded in a 6-well plate (Corning). After 24 h cells were treated with G1 or G36. Plates were incubated for at least seven days. As soon as colonies emerged, the plate was stained with crystal violet, scanned using Epson Scan 2 software (Epson, Suwa, Japan), and the colonies were analyzed using ImageJ 1.52a (Wayne Rasband).

Sphere formation. One thousand cells in 100 μl MEM per well were seeded in a 96-well ultra-low attachment plate with flat bottom (Corning). After 24 h, 100 μl of MEM including treatment was added. When SERPINE1-knockdown cells were used, the cells were seeded 24 h after transfection and 100 μl MEM without treatment was added after additional 24 h. The wells were photographed every 96 h using Celigo 2.1.0.96 software (Celigo, San Mateo, CA, USA). The tumor spheres were analyzed using ImageJ 1.52a (Wayne Rasband).

Western blot. Cells were lysed in cell lytic M buffer (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 0.1% phosphatase-inhibitor (C2978, Sigma-Aldrich) and 0.1% protease-inhibitor (P5726 and P8340, Sigma-Aldrich). Isolated proteins (40 μg) were fractioned using 12% sodium dodecyl sulfate (SDS) gel and electro-transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Primary antibodies used were rabbit anti-human SERPINE1/PAI-1 (ab31280, Abcam, Cambridge, UK) at 1:1,000 dilution and rabbit anti-human GAPDH (D16H11, Cell Signaling Technology, Danvers, MA, USA) at 1:2,000 dilution. The membrane was washed and incubated in horseradish peroxidase-conjugated donkey IgG anti-rabbit IgG (711-035-152, Dianova, Hamburg, Germany) at 1:10,000 dilution. Antibody-bond protein bands were assayed using ECL Chemostar (Intas, Göttingen, Germany).

Small interfering ribonucleic acid (siRNA) knockdown of SERPINE1. For reverse transfection of SiHa cells, a cell suspension with 147,000 cells/ml was centrifuged and the supernatant was aspirated. Afterwards, the cells were resuspended in a solution with 1.5 ml OptiMEM (Gibco Life Technologies, Carlsbad, CA, USA) per well. Meanwhile, the transfection mix for a 6-well plate, consisting of 500 μl OptiMEM, 3 μl SERPINE1 siRNA (sc-36179, Santa Cruz Biotechnology, Dallas, TX, USA) or control siRNA (sc-37007, Santa Cruz Biotechnology) and 5 μl Invitrogen Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) was vortexed and incubated for 20 min at room temperature. After incubation, the transfection mix was put in the wells with a total amount of 508 μl per well. The cell suspension was carefully added on top. The medium was changed after 6-12 h to MEM. The cells were either used 24 h after transfection for functional assays or three days after transfection for ribonucleic acid (RNA) isolation.

RNA isolation and cDNA synthesis. RNA from transfected cells was isolated using the FastGene RNA Basic Kit (FG-80250, Nippon Genetics Europe, Düren, Germany) according to the manufacturer’s instructions. RNA samples were diluted to a final concentration of 1 μg of RNA. Two microliters of 60 μM random primer (GeneOn, Ludwigshafen, Germany) and 1 μl deoxyribonucleotide triphosphates (dNTPs) (110-012, GeneOn) were added to every sample and the RNA was denatured by heating for 5 min to 70°C. After cooling the samples on ice, 4 μl of 10× reaction buffer (M0253L, New England Biolabs, Frankfurt, Germany), 1 μl M-MuLVRT reverse transcriptase (M0253L, New England Biolabs), 0.2 μl RNAse inhibitor (105-350, GeneOn) and 4.8 μl of diethyl pyrocarbonate (DEPC) (K028.1, Carl Roth, Karlsruhe, Germany) water [0.1% (v/v) DEPC in deionized water] were added and the mix was gently vortexed. cDNA was synthesized with an incubation step for 5 min at 25°C, a heating step for 1 h at 42°C and a denaturation step for 20 min at 65°C. The cDNA was then stored at −20°C.

RT-qPCR. cDNA samples were diluted to a concentration of 5 ng/μl with deionized water and 14 μl qPCR mix [2x; 75 mM Tris-HCl (pH 8.8), 20 mM (NH4)2SO4, 0.01% Tween 20, 3 mM MgCl2, 0.2 mM dNTPs, 0.25% Triton X-100, 20 U/ml Taq polymerase, 1:40,000 SYBR Green I, 500 mM Trehalose], 9 μl deionized water and 1 μl primer pair mix (10 μM) were added. The CFX Connect Optics module/RT-PCR and the CFX Connect Real-Time system (Bio-Rad) were used under following conditions: denaturation for 5 min at 95°C, 95°C for 10 s, cooling-down to 65°C for 30 s and heating up to 65-95°C. Step two and three were repeated 39 times. Gene expression was normalized to the housekeeping gene RPLP0 (forward primer 5′-GAT TGG CTA CCC AAC TGT TG-3′, reverse 5′-CAG GGG CAG CAG CCA CAA A-3′) and the samples were quantified based on a standard curve, existing of a 1:4 serial dilution of the cDNA. The SERPINE1 primers used were: forward 5′-ACC CTC AGC ATG TTC ATT GC-3′; reverse 5′-TCA TGT TGC CTT TCC AGT GG-3′.

Statistical analysis. All experiments in this study were performed in biological triplicates and technical duplicates at minimum. The results are presented as mean±SEM. One-way ANOVA, two-way ANOVA, and unpaired t-tests were performed in GraphPad Prism (v. 8.0.1, GraphPad Software Inc., San Diego, CA, USA). The one-way ANOVA analysis was followed by Dunnetts’s or Tukey’s multiple comparisons test. Unpaired, two-tailed, parametric t-tests were used under the assumption that both of the compared groups had the same standard deviation. Results were considered statistically significant when p<0.05.

Results

Effects of treatment of CSCC cells with GPER1 agonist G1 and antagonist G36 on cell migration. Treatment with GPER1 agonist G1 showed a concentration-dependent reduction in cell migration. The higher the G1 concentration, the slower the gap closed. When treated with 5 μM of G1, the gap area of SiHa CSCC cells was open on average to 76.4±5.20% after 30 h (n=4), while the control showed an average gap opening of 14.7±1.55% (p=0.0084, n=4; Figure 1A). Treatment of C33A CSCC cells with GPER1 agonist G1 showed similar effects, although statistical significance was reached after 45 h at the highest concentration (p=0.0285; Figure 1C). Treatment with G36 hardly made a difference compared to the control without treatment in both cell lines (Figure 1B, D).

Figure 1.
Figure 1.

Migration of cervical squamous cell carcinoma (CSCC) cells after treatment with GPER1 agonist G1 and antagonist G36. (A, C) Dose-dependent reduction of migration of SiHa (A) and C33A cells (C) in response to treatment with GPER1 agonist G1. Treatment of SiHA (B) and C33A (D) CSCC cells treated with GPER1 antagonist G36 showed no change in migration capacity. Two-way ANOVA with Dunnett’s multiple comparisons test, mean with standard error of the mean (SEM); n=4; *p<0.05, **p<0.01. The asterisks refer to statistically significant differences between the corresponding graph line and the ethanol-treated control.

Effects of treatment of CSCC cells with GPER1 agonist G1 and antagonist G36 on colony formation. After treatment of SiHa CSCC cells with 1 μM and 2.5 μM of GPER1 agonist G1, no significant changes in colony number were observed (Figure 2A). However, treatment with 5 μM of G1 was associated with a significantly decreased number of colonies compared to the control (674.0±110.9 vs. 1,322±37.7, n=3, p=0.0442; Figure 2A). In the same manner, higher concentrations of GPER1 agonist G1 had a statistically significant effect on the colony size of SiHa CSCC cells. Specifically, treatment with 2.5 μM or 5 μM G1 colony size was reduced to 24,600±7,136 px (p=0.0244, n=5) or to 13,991±2,865 px (p=0.0181, n=5), respectively, compared to the control (Figure 2B). Treatment with GPER1 antagonist G36 did not result in a significant change of colony number or size (Figure 2A, B).

Figure 2.
Figure 2.

Colony formation and size of cervical squamous cell carcinoma (CSCC) cells after treatment with GPER1 agonist G1 and antagonist G36. The number of colonies was decreased in a dose-dependent manner in SiHa (A) and C33A (C) cells under treatment with GPER1 agonist G1. Similar reduction was observed in the size of colonies in SiHa (B) and C33A (D) cells treated with GPER1 agonist G1. Treatment of the cells with GPER1 antagonist G36 showed no significant change in colony formation or size of colonies. The horizontal lines in the violin plots represent the median, and the first and third quartiles. Ordinary one-way ANOVA with Dunnett’s multiple comparisons test, mean with standard error of the mean (SEM); (A) n=3, (B-D) n=5; *p<0.05.

After treatment of C33A CSCC cells with 1 μM of GPER1 agonist G1, no significant changes in colony number were observed, while the concentrations of 2.5 μM and 5 μM of G1 were associated with a significantly decreased number of colonies to 618.8±72.4 (2.5 μM, p=0.0435, n=5) or to 316.8±81.9 (5 μM, p=0.0239, n=5) compared to control (3.291.0±1,180.4, n=5; Figure 2C). Treatment of C33A CSCC cells with 1 μM of GPER1 agonist G1 resulted in a significant decrease of colony size, compared to the control cells (33,792±13,334 px vs. 203,328±80,178 px, p=0.0366, n=5). Similar results were obtained after treatment with 2.5 μM or 5 μM G1 (2.5 μM: 2,778±450 px, p=0.0120, n=5; 5 μM: 4,501±1,502 px, p=0.0128; Figure 2D). Treatment with GPER1 antagonist G36 did not result in a significant change of colony number or colony size (Figure 2C, D).

Effects of treatment of CSCC cells with GPER1 agonist G1 and antagonist G36 on sphere formation. After treatment of SiHa CSCC cells with 1 μM of GPER1 agonist G1, a slight, though not statistically significant, decrease of formed spheres was observed compared to control (Figure 3A). After treatment of SiHa CSCC cells with 2.5 μM or 5 μM G1, no spheres were formed (2.5 μM, 0.0±0.0, p=0.0026, n=3; 5 μM, 0.0±0.0, p=0.0026, n=3; Figure 3A). A similar effect was demonstrated on the sphere size after treatment of SiHa CSCC cells with GPER1 agonist G1. A slight, non-significant decrease of sphere size was observed, compared to the control, when the lowest concentration (1 μM) of G1 was used (Figure 3B) and after treatment of SiHa cells with 2.5 μM or 5 μM G1, no sphere size could be measured as no spheres were formed (Figure 3B). Treatment with GPER1 antagonist G36 did not alter the sphere number or sphere size (Figure 3A, B).

Figure 3.
Figure 3.

Sphere formation and size of cervical squamous cell carcinoma (CSCC) cells after treatment with GPER1 agonist G1 and antagonist G36. (A, C) Number and size of formed spheres was significantly reduced under treatment with GPER1 agonist G1. (B, D) CSCC cells treated with GPER1 antagonist G36 show no significant change in number and size of formed spheres. The horizontal lines in the violin plots represent the median and the first and third quartiles. Ordinary one-way ANOVA with Dunnett’s multiple comparisons test, mean with standard error of the mean (SEM); n=3; **p<0.01, ****p<0.0001.

After treatment of C33A CSCC cells with 1 μM of GPER1 agonist G1, a significant decrease in number of formed spheres was observed compared to control (7.7±2.6 vs. 20.0±1.7, n=3, p=0.0014; Figure 3C). After treatment of C33A CSCC cells with 2.5 μM or 5 μM G1, no spheres were formed (Figure 3C). Regarding the sphere size, treatment of C33A CSCC cells with 1 μM of GPER1 agonist G1 resulted in a non-significant decrease (457,817±219,238 px vs. control 1,199,089±296,810 px, p=0.0369, n=3; Figure 3D). While, after treatment with 2.5 μM or 5 μM G1, no sphere size could be measured as no spheres were formed (2.5 μM, 0±0 px, p=0.0034, n=3; 5 μM, 0±0 px, p=0.0034, n=3; Figure 3D). Treatment with GPER1 antagonist G36 did not result in a significant change of sphere number or sphere size (Figure 3C, D).

Effects of treatment of CSCC cells with GPER1 agonist G1 and antagonist G36 on PAI-1 expression. Since C33A CSCC cells could not be successfully transfected with SERPINE1 siRNA, the following experiments were only performed with SiHa cells. Expression of the SERPINE1 gene product PAI-1 was first analyzed using Western blot analysis. The relative PAI-1 expression on SiHa CSCC cells was decreased, compared to control (0.4±0.09 vs. control=1.0, n=3, p=0.0031) after treatment with 2.5 μM GPER1 agonist G1 (Figure 4A). On the contrary, treatment of SiHa cells with 2.5 μM GPER1 antagonist G36 increased the relative PAI-1 expression (2.5±0.5 vs. control=1.0, n=3, p=0.0337; Figure 4A).

Figure 4.
Figure 4.

Relative expression of PAI-1 in SiHa cervical squamous cell carcinoma (CSCC) cells after treatment with GPER1 agonist G1 and antagonist G36 and effects of SERPINE1/PAI1 knockdown on viability and migration. (A) PAI-1 expression was significantly reduced after treatment with 0.5 μM GPER1 agonist G1 and significantly increased after treatment with 1 μM antagonist G36. (B) Successful knockdown of SERPINE1/PAI1 was confirmed by RT-qPCR. No impact of SERPINE1/PAI1 knockdown on viability (C) and migration (D) in SiHa CSCC cells was found. (A-C) Unpaired t-test, two tailed, mean with standard error of the mean (SEM); (A) n=3; (B) n=6, (C) n=4; *p<0.05, **p<0.01. (D) Two-way ANOVA with Dunnett’s multiple comparisons test, mean with standard error of the mean (SEM); n=4.

Effects of SERPINE1/PAI-1 suppression on CSCC cell viability and cell migration. To investigate the role of SERPINE1/PAI-1 in GPER1-mediated antitumor activity in CSCC, SERPINE1 expression was suppressed using siRNA. Successful suppression of SERPINE1 expression was confirmed by RT-qPCR (Figure 4B). After siRNA knockdown in SiHa CSCC cells, SERPINE1 mRNA relative expression was reduced, compared to the control (0.29±0.10 vs. control=1.0±0.13, p=0.0053, n=4). The viability of SiHa CSCC cells was hardly affected by suppression of SERPINE1 (0.88±0.10 vs. control=1.0, p=0.2588, n=6; Figure 4C). No changes in migration were observed after suppression of SERPINE1 in SiHa CSCC cells (Figure 4D).

Effects of SERPINE1/PAI-1 suppression on CSCC cell colony formation and sphere formation. After suppression of SERPINE1 expression in SiHa CSCC cells, the number of colonies formed was not altered (Figure 5A), whereas the colony size was slightly, non-significantly reduced compared to control (Figure 5B). Sphere formation of SiHa CSCC cells was significantly influenced by suppression of SERPINE1. Specifically, the number of spheres as well as the size of spheres were significantly decreased, compared to the control (number of spheres: 2.56±0.38 vs. 3.67±0.41, n=9, p=0.0314; size of spheres: 365,847±51,121 px vs. 512,321±39,889 px, n=9, p=0.0371; Figure 5C, D).

Figure 5.
Figure 5.

Colony and sphere formation and size of SiHa cervical squamous cell carcinoma (CSCC) cells after suppression of SERPINE1/PAI1 expression by siRNA. The number of colonies remained unchanged after knockdown of SERPINE1/PAI-1 (A), while the colony size was slightly reduced (B). Sphere number (C) and size (D) of SiHa CSCC cells was significantly reduced after SERPINE1/PAI1 knockdown. (A, C) Violin plot with median (middle line), first quartile (25%, lower line) and third quartile (75%, upper line). Unpaired t-test, two tailed (A, B, D), one tailed (C), mean with standard error of the mean (SEM); (A, B) n=11, (B-D) n=9; *p<0.05.

Discussion

SERPINE1/PAI-1 is an oncogene that has already shown its malignant properties in different tumor entities, such as gastric carcinoma (13). Recently a link between SERPINE1/PAI-1 and the G-protein coupled estrogen receptor 1 (GPER1) has been shown. Besides to the findings that GPER1 has tumor suppressive effects in CC, SERPINE1/PAI-1 was up-regulated after suppression of GPER1 expression (9).

Since the role of GPER1 in cancer is still controversial (15), this work aimed to examine the tumor suppressive effect of GPER1 in CSCC cell lines using GPER1 agonist G1 and antagonist G36. Although G1 is considered a selective GPER1 agonist (6), non-receptor-mediated effects have been established by showing that G1 interacts directly with tubulin and exerts a tumor suppressive effect on breast cancer cells even when GPER1 expression is inconstant (16). However, dose-response experiments using vulvar carcinoma cells revealed that the effect of G1 is via GPER1. More specifically, it was shown that the effect of G1 on cell proliferation was abolished in a concentration-dependent manner by GPER1 antagonist G36 (17).

In this work, the tumor suppressive effect of GPER1 on CSCC was confirmed in vitro, as shown by the suppressive effect of GPER1 agonist G1 on viability and migration of CSCC cells. In a previous study, it was demonstrated that knockdown of GPER1 leads to clear signs of epithelial-mesenchymal transition (EMT) and results in increased 3D invasion. However, this was shown in the CC cell line HeLa, which is derived from adenocarcinoma of the cervix (9). Previous publications have shown that GPER1 not only has different functions in different tumor entities, but also in different cell lines of the same tumor entity (18). Nevertheless, GPER1 mostly showed similar effects in the cell lines examined so far, which confirms the inhibitory effect of GPER1 on migration of CSCC cell lines.

Activation of GPER1 using agonist G1 had an inhibitory effect on clonogenicity of both CSCC cell lines. In contrast, blocking GPER1 with an antagonist had no effect on the ability to form colonies and spheres. However, increased stem cell properties, such as increased colony and tumor sphere formation, have been shown after knockdown of GPER1 in CC cells (9). In line with these results, it has already been shown that SiHa CSCC cells form larger spheres under GPER1 suppression (9).

Regarding the role of GPER1 in cancer, it has been shown that it is implicated in the activation of oncogenes. For instance, stimulation of GPER1 by G1 inhibits the up-regulation of the oncogene JUN in colorectal carcinoma (19). A direct link between GPER1 and the oncogene SERPINE1/PAI-1 has also been shown in CC. Ruckriegl et al. showed that SERPINE1/PAI-1 is up-regulated after GPER1 knockdown (9). A connection between GPER1 and SERPINE1/PAI-1 was also demonstrated in this study. Treatment with GPER1 agonist G1 led to a decreased expression of SERPINE1/PAI-1, while blockade of GPER1 with the antagonist G36 resulted in a significantly increased expression of SERPINE1/PAI-1 in SiHa CSCC cells.

Knockdown of SERPINE1/PAI-1 in SiHa-CSCC cells led to a reduced clonogenicity. However, cell migration remained unaffected. Unfortunately, successful suppression of SERPINE1/PAI-1 expression could not be achieved in C33A cells.

Based on the results to date, it can be assumed that SERPINE1/PAI-1 generally has a tumor-promoting effect and that suppression of SERPINE1/PAI-1 attenuates the tumorigenic properties of carcinoma cells. Pavón et al. have demonstrated that silencing of SERPINE1/PAI-1 resulted in inhibition of growth, migration, and invasion ability of the tumor cells in head and neck tumors (13). In colorectal cancer cells, SERPINE1/PAI-1 knockdown led to reduced viability of tumor cells (20). Yuan et al. also showed a correlation between the antineoplastic effect of SERPINE1/PAI-1 silencing and an increase in oxidative stress in the cells (20). According to these findings, the tumor cells would not only be exposed to SERPINE1/PAI-1 knockdown but also to increased oxidative stress, which could inhibit the tumor properties. Thus, even in CSCC cells with SERPINE1/PAI-1 knockdown, it is not yet possible to say conclusively whether tumorigenicity is reduced only by the lower expression of SERPINE1/PAI-1 or additionally by oxidative stress. Based on studies that showed higher SERPINE1/PAI-1 concentrations in metastatic melanoma and in triple-negative breast cancer compared to normal tissue (21), we suggest that SERPINE1/PAI-1 could also have an enhancing effect on metastasis in CSCC. In colorectal carcinoma, inhibition of SERPINE1/PAI-1 reduced migration and invasion (22). However, since SiHa CSCC cells showed no differences in cell migration after SERPINE1/PAI-1 knockdown, SERPINE1/PAI-1 may be less important for migration of CSCC cells.

A very recent study pointed to the potential of SEPRINE1 family genes as biomarkers for cancer prognosis (12). SERPINE1/PAI-1 could therefore be considered a potential target for anti-cancer therapies in CSCC. SERPINE2, which also belongs to the SERPINE family, is also a plasminogen activator inhibitor (23) and can provide some indications of tumor malignancy.

In renal cell carcinoma (RCC), silencing of SERPINE2 led to significantly suppressed growth and reduced invasion of RCC cells, while overexpression of SERPINE2 significantly promoted metastasis of RCC cells both in vitro and in vivo. The same group also showed that the different expression patterns of renal malignant cells play a significant role in the progression of RCC. Thus, SEPRINE2 may be a novel therapeutic target for the inhibition of metastasis in advanced renal carcinoma (24).

In CSCC, only the inhibitory effect of SERPINE1/PAI-1 suppression on sphere formation was significant, otherwise the effects were rather small. Numerous other processes and oncogenes likely play a decisive role in the malignancy of CSCC.

Experiments have shown that SERPINE2 contributes to the promotion of a tumor-supporting milieu and an immunosuppressive microenvironment in gastric cancer (25). Our research supports the idea that SERPINE1 has a tumor-promoting effect in CSCC possible through interaction with other cells, especially immune cells and that further experiments with immune cells would be useful. In addition, further trials with a SERPINE1/PAI-1 inhibitor could provide more information about the role of SERPINE1/PAI-1 in CSCC. Several PAI-1 inhibitors are already known. One example would be XR1853, which induces the transition from active PAI-1 to inactive PAI-1 (26). SERPINE1/PAI-1 is currently being discussed as a molecular marker for the prognosis of gastric carcinoma (27).

The conclusions of our study are limited by various aspects. The variability in CC and also in CSCC, when considered alone, is very high. For example, we had to disregard the HPV status when using only two cell lines. In order to generalize the results for CSCC or even CC as a whole, further extensive studies with additional CC or CSCC cell lines as well as in vivo studies must be carried out, particularly with regard to migration, invasion, and metastasis.

Conclusion

Activation of GPER1 in SiHa and C33A CSCC cells resulted in tumor suppressive effects and reduced clonogenicity and migration. GPER1 regulated expression of SERPINE1/PAI-1 in SiHa and C33A CSCC cells. Activation of GPER1 led to decreased SERPINE1/PAI-1 expression, while inhibition of GPER1 promoted SERPINE1/PAI-1 expression. Knockdown of SERPINE1/PAI-1 in SiHa CSCC cells decreased tumorigenic potential but not migration. Therefore, SERPINE1/PAI-1 does not appear to be the decisive factor for GPER1-regulated SiHa CSCC cell migration.

Since SERPINE1/PAI-1 showed an influence on CSCC cells, but this influence was not constant, the use of SERPINE1/PAI-1 alone as a biomarker would probably not be effective. Nevertheless, GPER1 may be a suitable target for SERPINE1/PAI-1 suppression in CSCC in the future.

Acknowledgements

The Authors thank Sonja Blume and Matthias Läsche for excellent technical assistance.

Footnotes

  • Conflicts of Interest

    The Authors declare that they have no competing interests.

  • Authors’ Contributions

    Conceptualization, C.G.; investigation, L.R. and S.R.; writing, original draft preparation, L.R. and C.G.; writing, review and editing, J.G.; project administration, C.G. All Authors have read and agreed to the published version of the manuscript.

  • Received August 22, 2024.
  • Revision received September 18, 2024.
  • Accepted September 30, 2024.

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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G Protein-coupled Estrogen Receptor 1 (GPER1) Regulates Expression of SERPINE1/PAI-1 and Inhibits Tumorigenic Potential of Cervical Squamous Cell Carcinoma Cells In Vitro
LINEA RÖRIG, SOPHIA RUCKRIEGL, JULIA GALLWAS, CARSTEN GRÜNDKER
Cancer Genomics & Proteomics Jan 2025, 22 (1) 13-23; DOI: 10.21873/cgp.20482
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