β-catenin signaling inhibitors ICG-001 and C-82 improve fibrosis in preclinical models of endometriosis

Endometriosis exhibits unique characteristics, such as fibrosis, resistance to apoptosis, and promotion of cell proliferation; however, its pathophysiology is not fully understood. Recurrence rates after treatment are high, and the progression risk continues until menopause; hence, more effective therapy for endometriosis is needed. CREB-binding protein (CBP)/β-catenin signaling inhibitors have demonstrated antifibrogenetic effects in liver, lung, and skin diseases. The present study evaluated the effects of two CBP/β-catenin signaling inhibitors, ICG-001 and C-82, on the progression of endometriosis using endometriotic cyst stromal cells from the ovary and normal endometrial stromal cells from the uterus. ICG-001 was also evaluated in a mouse model. ICG-001 and C-82 inhibited cell proliferation, fibrogenesis, and cell migration, and promoted apoptosis in vitro. ICG-001 inhibited the growth of endometriotic lesions in the mouse model. CBP/β-catenin signaling plays an important role in the pathophysiology of endometriosis. Inhibiting the CBP/β-catenin signal can be a therapeutic target for endometriosis.

Scratch assay. In a scratch assay to evaluate cell migration (Fig. 3A), the untreated control ECSC migrated and became confluent 24 hours after the scratch. ICG-001 and C-82 inhibited cell migration (Fig. 3A). Migration distances were calculated and defined as 100% in the control group. Figure 3B shows a significant decrease in cell migration by 64% with ICG-001 and by 54% with C-82 in ECSC (p = 0.0168, p = 0.0212).
Gel contractility. Three-dimensional collagen gel cultures of ECSC after treatment with the CBP/β-catenin signaling inhibitors showed significantly inhibited contractility by ICG-001 and C-82 as compared with the untreated control. The surface area was calculated and defined 100% in the control group. The ICG-001 group was 286% and the C-82 group was 662% in ECSC ( Fig. 3C,D, p = 0.0001, p = 0.0063).
α-SMA expression. The expression of alpha-smooth muscle actin (α-SMA) mRNA was significantly higher in ECSC than in NESC (Fig. 4A, p = 0.0018). Treatment with ICG-001 and C-82 significantly downregulated α-SMA mRNA expression in ECSC (Fig. 4B, p = 0.0000, p = 0.0000). The protein expression of α-SMA as examined by western blot analysis was significantly higher in ECSC than in NESC (Fig. 4C,D, p = 0.0400), but neither ICG-001 nor C-82 resulted in a significant change in the protein expression of α-SMA in ECSC (Fig. 4E,F, p = 0.7381, p = 0.8958). Endometriosis in mice model. In the experiments using a mouse model of endometriosis, we first confirmed endometriotic lesions having been formed in the mouse abdomen 1 week after uterine implantation (Table S1) and then evaluated the therapeutic effects of ICG-001 in these mice (n = 10/group, x 4 groups). One mouse in the ICG-001 10 mg/kg group and one mouse in the ICG-001 100 mg/kg group died about 3 weeks after uterine implantation. After examining the mice by dissection, the cause of death was assumed to be excess bleeding caused by injections. The weights of the mice in the four groups were not significantly different (Fig. S1). All groups were confirmed to have endometriotic lesions by HE staining (Fig. S2A-D). Figure 5A shows a representative picture of an intraabdominal endometriotic lesion. The mean number of endometriotic lesions in the untreated group was significantly higher than those in all the ICG-001-treated groups (Fig. 5B, p = 0.0102, p < 0.001, p < 0.001). The total weight of the endometriotic lesions was highest in the untreated group and lowest in the ICG-001 100 mg/kg group (Fig. S3). The fibrosis of endometriosis was assessed immunohistochemically by modified Masson's staining (Fig. 5C), Sirius red staining (Fig. 5D), and α-SMA staining (Fig. 5E). We calculated the intensity of the collagen fibers in the modified Masson's staining using a Keyence BZ-9000 (Keyence, Chicago,  www.nature.com/scientificreports www.nature.com/scientificreports/ IL, USA). The collagen fibers (aniline blue stained area) were significantly reduced in the ICG-001 50 mg/kg and 100 mg/kg groups (Fig. 5F, p = 0.0012, p < 0.001). α-SMA was observed in endometrial stromal lesions and its staining intensity was the weakest in the ICG-001 100 mg/kg group (Fig. 5E).

Discussion
The Wnt/β-catenin signaling pathway is involved in normal reproduction and development, as well as maintenance of normal physiological functions. The Wnt/β-catenin signaling pathway is further involved in progression of diseases by regulating cell proliferation, migration, invasion, and fibrogenesis 11,12,16,17 . Wnt/β-catenin signaling pathway inhibitors have been studied as potential treatments for various proliferative or fibrotic diseases, including cancers, liver cirrhosis or pulmonary fibrosis, and some inhibitors are currently being tested in clinical trials 8,17 . Although the antifibrotic effects of CBP/β-catenin inhibitors on lung fibrosis, liver fibrosis, and cancers have been examined 6,9,11,18,19 , their antifibrotic effect in endometriosis have not been investigated. Because activation of the Wnt/β-catenin signaling pathway in endometriosis is associated with fibrosis, cell proliferation, and resistance to apoptosis 5,20-22 , the inhibition of this pathway is recognized as a promising strategy for treating endometriosis. ICG-001 is a selective low molecular-weight inhibitor, and downregulates the expression of a subset of www.nature.com/scientificreports www.nature.com/scientificreports/ β-catenin/TCF-responsive genes 11 . ICG-001 inhibits β-catenin/TCF signaling by specifically binding to the cyclic AMP response element-binding protein CREBBP (CBP), thereby disrupting the CBP/catenin interaction 23 . PRI-724 is a second generation specific CBP/catenin antagonist 24 .
In this study, we tested a hypothesis that Wnt/β-catenin signaling inhibitors ICG-001 and C-82 have an antifibrotic effect in the preclinical models of endometriosis that had been validated by our group for evaluation of fibrosis 13,14,15 . The present study revealed significant upregulation of β-catenin protein expression in ECSC, suggesting activation of Wnt/β-catenin signaling pathway in the pathophysiology of endometriosis. ICG-001 and C-82 inhibited in vitro cell growth, cell migration, and fibrosis, and promoted apoptosis. The mRNA expression of α-SMA with ICG-001 and C-82 was significantly downregulated in ECSC, but there was no difference in protein expression (Fig. 4BE,F). It is believed that there is a time lag between the expression of mRNA and the expression of the protein. It is not expected that induced transcription immediately leads to increased protein levels because there is a delay to mature, export, and translate mRNA 25 .
Treatment with ICG-001 (10, 50, and 100 mg/kg) appeared to be nontoxic to mice, but effectively modified disease progression in the murine model of endometriosis where ICG-001 dose-dependently inhibited intra-abdomen fibrosis as evaluated. However, the number of endometriotic lesions, intensity of the modified Masson's staining, and the staining intensity of α-SMA were the weakest in the ICG-001 100 mg/kg group. Thus, further study is needed. Data generated from this study provide additional evidence to support the inhibition of the Wnt/β-catenin signaling pathway being an attractive therapeutic target to improve fibrosis and to reverse endometriotic lesions.
Prior to the present study, the relationship between CBP/β-catenin binding and endometriosis had not been fully examined, although there are some reports illustrating that the Wnt/β-catenin pathway is activated in endometriosis. Matsuzaki et al. demonstrated that fibrosis, cell proliferation, and cell migration were inhibited in ECSC 5,22,26 after blocking the Wnt/β-catenin pathway. The antifibrotic effect of CGP049040, a small molecule TCF/β-catenin antagonist, on fibrosis was shown in their mouse model of endometriosis. The staining scores by Sirius red and Masson trichrome stains were significantly lower in CGP049040 treated mice. Their data support our view that activation of the Wnt/β-catenin signaling pathway in endometriosis is associated with fibrosis. Although Matsuzaki et al. showed a reduction of collagen fiber by a small molecule antagonist for the TCF/β-catenin complex, they did not provide data on the reduction in size or the number of endometriotic lesions, whereas our study clearly demonstrated a reduction of endometriotic lesions in size and number with CBP/β-catenin inhibitors. CBP/β-catenin interaction is crucial in the pathological mechanism of endometriosis progression.
There are some limitations in the current study. Although no apparent drug-related toxicity was observed from the intraperitoneal findings or body weight, the effects on other organs have not been confirmed. There is no denying that this drug may affect the functions or morphology of other organs. PRI-724 has been administrated to humans, and a clinical trial of PRI-724 (C-82) revealed that most of the observed side effects to humans were mild, such as reaction at the injection site, nausea, and constipation 8 . A high dose of ICG-001 was given intraperitoneally, and this dose may not be comparable to the clinical dose. In this study, the pharmacokinetics of ICG-001 were not evaluated; therefore, it is necessary to investigate the relationship between exposure and pharmacodynamics. The present endometriosis model was created by transplanting the uterine tissue of a normal mouse into the abdominal cavity of another mouse. It may not accurately reflect the condition of human endometriosis and its translationability is unknown. The menstrual cycle was different from what naturally occurs in the model mice because estradiol was continuously administered. Although this was translational research at the stage of confirming safety, there is a history of mice models being widely used for endometriosis studies in general, and it has been validated as a model that shows the dynamics of endometriosis.
In conclusion, we have identified the CBP/β-catenin signaling inhibitors, ICG-001 and C-82, as leads to promising therapeutic agents for the treatment of endometriosis. The CBP/β-catenin inhibitors suppressed cell migration and cell proliferation and promoted apoptosis. Hence, our data suggest that CBP/β-catenin inhibitors have a possibility not only to prevent but reduce fibrosis in endometriosis. Future studies will need to focus on searching and synthesizing more effective CBP/β-catenin inhibitors. The promotion of research and practical application of new drugs for endometriosis is important, and a clinical trial of CBP/β-catenin inhibitors for endometriosis should be planned for the near future.

Methods
Cell culture and reagents. Ovarian endometriosis tissues were obtained from patients with regular menstrual cycles who had undergone salpingo-oophorectomy or cystectomy for the treatment of ovarian endometriotic cysts (11 patients, aged 26-49 years). Normal endometrium tissues were obtained from patients who had undergone hysterectomy because of myoma or other benign diseases without endometriosis (6 patients, aged 43-45 years). None of the patients had received hormonal treatments for at least 6 months before surgery. All specimens were confirmed to be in the proliferative phases based on pathological observation, the menstrual cycle, or both. ECSC and NESC were isolated from the ovarian endometriotic tissues and normal uterine tissues using enzymatic digestion with collagenase. They were then confirmed to be stromal cells from the endometrium as previously described 27,28,29 . In addition, we performed basic experiments using western blotting. CD10 and vimentin were positive, and we confirmed the cells were derived from endometrial stroma. Isolated cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Nissui, Tokyo, Japan) supplemented with 100 IU/mL penicillin (Meiji Seika, Tokyo, Japan), 50 mg/mL streptomycin (Meiji Seika, Tokyo, Japan), and 10% heat-inactivated fetal bovine serum (FBS, all obtained from EQUITECH-BIO, Inc., Kerrville, Texas, USA) at 37 °C in air containing with 5% CO 2 . ECSC and NESC were confirmed to have >99% purity by immunocytochemical staining with vimentin (Santa Cruz Biotechnology, Dallas, USA), CD10 (Abcam, Cambridge, UK), cytokeratin (Bioss, Massachusetts, USA), and factor VIII (Novus Biologicals, Colorado, USA) after two to three passages. Cells from two to five passages were used. ICG-001 was synthesized in our laboratory and C-82 was provided by Prism Pharma Co., Ltd., Tokyo, Japan (Fig. S4). We have data that C-82 inhibits the binding between β-catenin www.nature.com/scientificreports www.nature.com/scientificreports/ and CBP 10 times more than ICG-001. The concentration used for acute lymphoblastic leukemia 23 was the reference for this study.
This study was approved by the Institutional Review Board of the Faculty of Medicine, Oita University (no. P16-01), and written informed consent was obtained from all patients. All methods involving patients were performed in accordance with relevant guidelines and regulations.
Immunohistochemical staining. Immunohistochemical staining (using the streptavidin-biotin-peroxidase method) was performed with mouse monoclonal antibody against human β-catenin (1:100 dilution; Cell Signaling #8480, Massachusetts, USA), and biotinylated goat anti-rabbit IgG (Dako, Tokyo, Japan). The staining intensities and sites of β-catenin in the endometriotic cysts of the ovaries compared with normal endometrial tissue were assessed.
Assessment of protein expression. The protein levels of β-catenin and α-SMA in ECSC and NESC were evaluated by western blot analysis. Antibodies against β-catenin (Cell Signaling, MA, USA), α-SMA (Gene Tex, Irvine, California, USA), and GAPDH (FUJIFILM Wako Chemical Corporation, Osaka, Japan) were used. Total protein from ECSC cells that were treated with ICG-001 (20 µM) or C-82 (2 µM) for 48 hours was extracted. The expression of each protein relative to the GAPDH protein in ECSC and NESC was analyzed using Image Lab software (Bio-Rad Laboratories, Hercules, CA, USA). The data were presented as the percentage of the values obtained for ECSC compared with NESC.

Assessment of apoptosis.
We determined the apoptosis levels of ECSC following ICG-001 or C-82 treatment by direct determination of nucleosomal DNA fragmentation using ELISA (Cell Death Detection ELISA, Roche Diagnostics, Basel, Switzerland) as Hirakawa et al. 2 described. ECSC (5 × 10 3 cells/well) were seeded in a 96-well flat-bottom microplate (Corning 96-well, Corning, NY, USA). After 24 hours, the culture medium was replaced with medium containing ICG-001 (0, 0.2, 2, 20, and 200 µM) or C-82 (0, 0.02, 0.2, 2, and 20 µM), and the cells were incubated for 24 hours. Then, the cells were lysed and centrifuged at 200 × g for 10 minutes, and the mono-and oligo-nucleosomes in the supernatants were quantified using an antihistone-biotin antibody (Nichirei Biosciences Inc., Tokyo, Japan). The concentration of the nucleosome-antibody complex was determined by measuring the absorbance at 405 nm using 2,2′-azino-di Assessment of cell migration. The scratch assay was used to assess cell migration and proliferation. ECSC were incubated in 12-well culture plates. When cells were 80% confluent, each plate was scratched with the same straight line. The medium was replaced with medium containing ICG-001 (20 µM) or C-82 (2 µM). The concentrations were determined from the results of the cell proliferation assay and the assay of the anti-apoptotic effect. Photos were taken on a microscope and the gap distances were quantitatively evaluated using BZ9000 (Keyence Corp., Osaka, Japan) at 0 and 24 hours after the scratch. Narrow distance between scratch edges means great migration ability and strong cell growth. The ratio before and after migration was calculated. A migration ratio of 100% indicates the highest migration ability. www.nature.com/scientificreports www.nature.com/scientificreports/ Assessment of ECSC contractility. We have established a three-dimensional collagen gel culture system with ECSC as a model of fibrosis formation in endometriosis 2,29 . In this system, ECSC are cultured in floating collagen lattices to induce the reorganization and compaction of collagen fibers, resulting in the contraction of collagen gels. This culture system provides models of mechanically relaxed tissue with low tensile strength and the early developmental stage of endometriotic lesions with high tensile strength. Collagen gel contraction assays were performed as previously described 29 . Forty-eight hours after ICG-001 (20 µM) or C-82 (2 µM) treatment, the ECSC were embedded in the collagen gel (Cellmatrix type I-A; Nitta Gelatin, Osaka, Japan) and cultured three-dimensionally for a further 72 hours. ICG-001 (20 µM) or C-82 (2 µM) was also added to the culture medium. The collagen gels were then photographed and the area of the gel surface was measured with ChemiDoc XRS+ with Image Lab Software (Bio-Rad Laboratories, Hercules, California, USA).
Assessment of mRNA expression. Total RNA of ECSC and NESC was extracted using a miRNeasy Mini kit (Qiagen, Düsseldorf, Germany) following the manufacturer's instructions. In addition, total RNA from ECSC that were treated with ICG-001 (20 µM) or C-82 (2 µM) for 48 hours was extracted similarly to analyze the expression of α-SMA. Subsequently, cDNA was synthesized from 1 μg of total RNA using the Reverse Transcription System (Promega, Madison, WI, USA). Quantitative RT-PCR was carried out with a LightCycler 480 (Roche Diagnostics, Rotkreuz, Schweiz) using TaqMan Universal PCR Master Mix II with the following specific primers (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA): β-catenin (Assay ID: Hs00355045_ m1); α-SMA (Assay ID: Hs04406862_m1); glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Assay ID: Hs02786624_g1) as Hirakawa et al. 2 reported. The expression level of mRNA relative to GAPDH mRNA was calculated by the relative standard curve method. Standard curves for relative quantification were prepared. Quantity is expressed relative to an untreated control. The data from ECSC were presented as the percentage of the values compared with NESC.
In addition, three mice were examined for endometriotic lesions 1 week after implantation, and an additional 44 mice were prepared as uterus donors. Mice were anesthetized with 2% isoflurane and, after bilateral dorsal incision, the bilateral ovaries were resected. The incision was sutured with 4-0 nylon. Estradiol (1 μg/mouse, Fuji Pharma, Tokyo, Japan, diluted with corn oil) was injected intraperitoneally into the left side of the abdomen once a week after the ovariectomy 30,31 . Two weeks after the ovariectomy, donor mice were euthanized, the uterus was removed, minced, and injected peritoneally with Corning Matrigel into the recipient mice. The uterus from each donor mouse was divided into two parts and implanted into two recipient mice. One week after uterine implantation, the sites and numbers of endometriotic lesions in the abdomen were examined. After confirming implantation of endometriosis, we started drug administration. Intraperitoneal injections (ICG-001 0, 10, 50, 100 mg/kg, diluted with PBS) to the right side of the abdomen were administered three times a week for 1 week after the implantation. Six weeks after ICG-001 administration, mice were euthanized and dissected to examine the abdomen. The sites of implantation and the numbers and weights of endometriotic lesions were recorded. An experimental assistant who was blinded to this experiment measured each resected specimen with an electronic analytical scale (SHIMADZU AG 204). Resected endometriotic lesions and uteruses were embedded in paraffin and stained by HE and anti-α-SMA antibody (Gene Tex, Irvine, California, USA). The histological tissue of the endometriotic lesions was also observed with modified Masson's staining (Scy Tek Laboratories, Utah, USA) and Sirius red staining (Polysciences, Inc., PA, USA) to evaluate fibrosis. To assess the extent of fibrosis, we analyzed the stained areas of collagen fiber by modified Masson's staining using BZ9000 (Keyence Corp., Osaka, Japan). The analysis area was defined by the detection of both endometrial epithelial cells and endometrial stromal cells under high-power magnification.
Statistics. The data were analyzed and presented as percentages relative to the corresponding control values as mean ± standard deviation. All statistical analyses were performed using the Excel statistical software package (BellCurve for Excel, version 2.15; Social Survey Research Information Co., Ltd., Tokyo, Japan). Significance was set at p < 0.05 with the Bonferroni correction and Student's t-test. Study approval. This study was approved by the Institutional Review Board of the Faculty of Medicine, Oita University (No. P16-01), and written informed consent was obtained from all patients. All methods involving humans were performed in accordance with relevant guidelines and regulations. The mouse model study was approved by Oita University Animal Ethics Committee (No. 172901). We followed the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.