Therapeutic Effects of In Vivo Administration of An Inhibitor of Tryptophan 2,3-dioxygenase (680C91) for the Treatment of Fibroids: A Preclinical Study

Abstract

Objective:

Fibroids are characterized by marked overexpression of Tryptophan 2,3 dioxygenase (TDO2). The objective of this study was to determine the effectiveness of in vivo administration of an inhibitor of TDO2 (680C91) on fibroid size and gene expression.

Design:

Animal and ex vivo human study.

Subjects:

SCID mice bearing human fibroid xenografts treated with vehicle and TDO2 inhibitor.

Intervention:

Daily intraperitoneal administration of 680C91 or vehicle for two months and in vitro studies with fibroid explants.

Main Outcome Measures:

Tumor weight and gene expression profile of xenografts and in vitro mechanistic experiments using fibroid explants.

Results:

Compound 680C91 was well-tolerated with no effects on blood chemistry and body weight. Treatment of mice with 680C91 resulted in 30% reduction in the weight of fibroid xenografts after two months of treatment and as expected lower levels of kynurenine, the byproduct of tryptophan degradation and an endogenous ligand of AhR (aryl hydrocarbon receptor) in the xenografts. The expression of CYP1B1 (cytochrome P450 family 1 subfamily B member 1), TGF-β3 (transforming growth factor beta 3), FN1 (fibronectin), CDK2 (cyclin-dependent kinase 2), E2F1 (E2F transcription factor 1), IL-8 (interleukin 8) and SPARC (secreted protein acidic and cysteine rich) mRNA, were lower in the xenografts of mice treated with 680C91 as compared with vehicle controls. Similarly, the protein abundance of collagen, FN1, CYP1B1 and SPARC were lower in the xenografts of 680C91 treated mice as compared with vehicle controls. Immunohistochemical analysis of xenografts indicated decreased expression of collagen, Ki67 and E2F1 but no significant changes in cleaved caspase 3 expression in mice treated with 680C91. The levels of kynurenine in the xenografts showed a direct correlation with the tumor weight and FN1 levels. In vitro studies with fibroid explants showed a significant induction of CYP1B1, TGF-β3, FN1, CDK2, E2F1, IL8 and SPARC mRNA by tryptophan which could be blocked by co-treatment with 680C91 and the AhR antagonist CH-223191.

Conclusion:

The results indicate that correction of aberrant tryptophan catabolism in fibroids could be an effective treatment through its effect to reduce cell proliferation and ECM accumulation.

Capsule:

Correction of dysregulated tryptophan catabolism could be an effective treatment of fibroids

Introduction

Uterine fibroids are common benign uterine smooth muscle tumors, affecting up to 40-70% of reproductive-aged women (1–3). These tumors can cause abdominal and pelvic pain, excessive menstrual bleeding, and infertility (1–3). The mechanism underlying the pathogenesis of fibroids is not fully understood and the subject of intense investigation. Recently our group reported for the first time a marked dysregulation of tryptophan degradation pathway with marked overexpression of TDO2 and IDO1 (indoleamine 2,3-dioxygenase 1) mRNA and protein in fibroids as compared with their matched myometrium (4, 5). These enzymes catalyze the first step in the degradation of tryptophan to N-formylkynurenine which is then hydrolyzed to kynurenine by NFK (N-formyl-L-kynurenine) formamidase (6). The levels of kynurenine are a reflection of TDO2/IDO1 enzyme activity and as would be expected we showed fibroids had higher kynurenine levels as compared with myometrium (4, 5). We also found that race and the presence of MED12 (Mediator Complex Subunit 12) mutation in the tumor positively influenced TDO2 but not IDO1 expression (4). In the same publication we reported that treatment of LSMC (leiomyoma smooth muscle cells) and MSMC (myometrial smooth muscle cells) spheroids with the TDO2 inhibitor, 680C91 but not the IDO1 inhibitor, Epacodostat significantly repressed cell proliferation and the expression of collagen type I (COL1A1) and type III (COL3A1) in a dose-dependent manner; these effects were more pronounced in LSMC spheroids as compared with MSMC spheroids. These results suggested that TDO2 is the enzyme with greater significance than IDO1 in fibroid pathogenesis. In a subsequent publication we reported that fibroids are characterized by dysregulation of a number of other enzymes in the tryptophan degradation pathway, with overexpression of TPH1 (tryptophan hydroxylase 1), KAT2 (potassium channel in arabidopsis thaliana 2), SLC7A8 (solute carrier family 7 member 8) and SLC7A5 (solute carrier family 7 member 5) mRNA and reduced expression of KYNU (kynureninase), WARS1 (tryptophanyl-tRNA synthetase 1) mRNA and no changes in the expression of WARS2 (tryptophanyl tRNA synthetase 2, mitochondrial), AFMID (arylformamidase), KMO (kynurenine 3-monooxygenase) and KAT1 (kynurenine aminotransferase 1), KAT3 (kynurenine aminotransferase III) and KAT4 (kynurenine aminotransferase IV) as compared with matched myometrium (5). Because kynurenine is a ligand of AhR we measured the expression of CYP1B1, a marker of AhR activation and showed these levels to be higher in fibroids as compared with myometrium (5). Another group has also reported on overexpression of TDO2 in fibroids (7). Based on our published in vitro data with compound 680C91 a specific TDO2 inhibitor on fibroid extracellular matrix (ECM) components and cellular proliferation we hypothesized that inhibition of TDO2 in vivo will have beneficial therapeutic effects for fibroids by reducing collagen expression and inhibiting cell proliferation, thereby causing shrinkage of tumors. To test this hypothesis, we used a mouse model for fibroids in which SCID (severe combined immunodeficiency) mice bearing human fibroid xenografts were treated with compound 680C91 or vehicle for two months.

Materials and Methods

Fibroid Specimens Collection

To reduce the variance among fibroids, tumors between 2 cm and 5 cm in diameter and with intramural location (n=12) were obtained from premenopausal patients undergoing hysterectomy for symptomatic fibroids at the Harbor-UCLA Medical Center. Endometrial hyperplasia and cancer were exclusionary criteria. Prior approval from the Institutional Review Board (18CR-31752-01R) at the Lundquist Institute was obtained. Informed consent was obtained from all the patients participating in the study who were not taking any hormonal medications for at least 3 months prior to surgery. The fresh tissues were used for cell isolation and for xenografts in the animal model, and the remaining tissues were snap-frozen and stored in liquid nitrogen for further analysis as previously described (8, 9).

Fibroid Animal Model

The protocol (32133-01) was approved by the IACUC at the Lundquist Institute at the Harbor-UCLA Medical Center. Female ovariectomized SCID/Beige mice (Charles River Laboratories, Hollister, CA, USA) 9–12 weeks old were implanted with pellets (Innovative Research of America, Sarasota, FL, USA) containing estradiol (0.075 mg/60 d release) and progesterone (75 mg/60 d release) as previously reported (10, 11). A portion of fresh fibroid weighing 0.5 g was cut aseptically into 5-10 pieces using a razor blade and implanted in the flank of a mouse. 12 pairs of explants were implanted in 24 mice with half receiving 680C91 and the other 12 mice receiving the vehicle, thus allowing comparison of each treated tumor to its own control. After 3 days of recovery, mice were injected daily with the vehicle or 680C91 (10 mg/Kg). The dose of 680C91 used was based on a prior publication examining its efficacy in a mouse model of chronic asthma (12). 680C91 (Cayman Chemical, Ann Arbor, Michigan, USA) was dissolved with DMSO (0.05 mg/ul), then diluted with a vehicle buffer (DMSO:saline=11:9) for intraperitoneal (i.p.) administration (total injection of 100 ul volume per mouse). After two months of treatment animals were sacrificed, and blood was obtained by cardiac puncture. Tumor explants were carefully dissected free of surrounding tissue and then were weighed and frozen. Animals were weighed before and after treatment with 680C91 or the vehicle.

Blood Chemistry Panel

We analyzed plasma chemistry after 8 weeks of treatment with the vehicle or 680C91. The plasma (100 μL) was pipetted into the Comprehensive Diagnostic Profile Rotor (#500-1038, Abaxis, Union City, CA, USA), and the values of glucose, creatinine, BUN, phosphorus, sodium, albumin, alkaline phosphatase, serum glutamic pyruvic transaminase (SGPT; ALT), total protein, globulin, total bilirubin, and amylase were analyzed using the VetScan VS2 chemistry analyzer (Abaxis, Union City, CA, USA).

Quantitative RT-PCR

Total RNA was extracted from the xenografts using TRIzol (Thermo Fisher Scientific Inc., Waltham, MA, USA). RNA concentration and integrity were determined using a Nanodrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) as previously described (13). Briefly, 2 μg of RNA was reverse transcribed using random primers for selected genes according to the manufacturer’s guidelines (Applied Biosystems, Carlsbad, CA, USA). Quantitative RT-PCR was carried out using the SYBR gene expression master mix (Applied Biosystems). Reactions were incubated for 10 min at 95 °C followed by 40 cycles for 15 s at 95 °C and 1 min at 60 °C. The expression levels of selected genes were quantified using Invitrogen StepOne System, with FBXW2 (F-box and WD repeat domain containing 2) used for normalization (14). All reactions were run in triplicate, and relative mRNA expression was determined using the comparative cycle threshold method (2-ΔΔCt), as recommended by the supplier (Applied Biosystems). Values were expressed as fold change compared to the control group. The primer sequences used were as follows: CYP1B1 (sense 5’-GGCCACTATCACTGACATCTTC-3’, antisense 5’-CGAGTCTGCACATCAGGATAC-3’); FN1 (sense 5’-ACCGAAATCACAGCCAGTAG-3’, antisense 5’-CCTCCTCACTCAGCTCATATTC-3’); CDK2 (sense, 5’-TTCCCCTCATCAAGAGCTATCTGT-3’; antisense, 5’-ACCCGATGAGAATGGCAGAA-3’); E2F1 (sense 5’-GGACTCTTCGGAGAACTTTCAGATC-3’, antisense 5’- GGGCACAGGAAAACATCGA-3’); TGF-β3 (sense 5’-CGGGCTTTGGACACCAATTA-3’, antisense 5’-GGGCGCACACAGCAGTTC-3’); IL8 (sense, 5’-CTTGGCAGCCTTCCTGATTT-3’; antisense, 5’-TTCTTTAGCACTCCTTGGCAAAA-3’); SPARC (sense, 5’-TACATCGGGCCTTGCAAATA-3’; antisense, 5’-TGTCCTCATCCCTCTCATACA-3’); and FBXW2 (sense 5’- CCTCGTCTCTAAACAGTGGAATAA-3’, antisense 5’- GCGTCCTGAACAGAATCATCTA-3’).

Immunoblotting

The total protein isolated from xenografts of the vehicle and 680C91 groups was subjected to immunoblotting as previously described (15, 16). Briefly, samples were suspended in a radioimmunoprecipitation assay (RIPA) buffer containing 1 mM EDTA (ethylenediamine tetra acetic acid) and EGTA (ethylene glycol tetra acetic acid) (Boston BioProducts, Milford, MA, USA) supplemented with 1 mM PMSF (phenylmethylsulfonyl fluoride) and a complete protease inhibitor mixture (Roche Diagnostics, Indianapolis, IN, USA), sonicated, and centrifuged at 4 °C for 10 min at 14,000 rpm. The concentration of protein was determined using the BCA^™ Protein Assay Kit (Thermo Scientific Pierce, Waltham, MA, USA). Equal aliquots (30 micrograms) of total protein for each sample were denatured with SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) sample buffer and separated by electrophoresis on an SDS polyacrylamide gel. After transferring the samples to a nitrocellulose membrane, the membrane was blocked with TBS-Tween + 5% milk and probed with the following primary antibodies: CYP1B1, FN1 and SPARC (Proteintech Group, Inc., Chicago, IL, USA). The membranes were washed with TBS containing 0.1% Tween-20 wash buffer after each antibody incubation cycle. SuperSignal West Pico Chemiluminescent Substrate^™ (Thermo Scientific Pierce, Waltham, MA, USA) was used for detection, and photographic emulsion was used to identify the protein bands, which were subsequently quantified by densitometry. The densities of the specific protein bands were determined using the image J program (http://imagej.nih.gov/ij/), normalized to a band obtained from staining the membrane with Ponceau S. Results were expressed as means ± SEM as a ratio relative to the control group, designated as 1.

Measurement of Kynurenine

Kynurenine concentration in paired leiomyoma and myometrium homogenates (n=12) was measured in duplicate using the Human Kynurenine ELISA kit (MBS766153; MyBioSource) according to the manufacturer’s instructions. Absorbance of each plate was measured spectrophotometrically at a wavelength of 450 nm and the concentration was determined by comparing the optical density value of samples to the standard curve.

Enzyme-Linked Immunosorbent Assay

The total collagen content in xenografts of vehicle and 680C91 treated groups (n=12) was measured in duplicate using the QuickZyme Total Collagen Assay Kit (QuickZyme Biosciences, Leiden, Netherlands) according to the manufacturer’s instructions. The absorbance of each plate was measured spectrophotometrically at a wavelength of 570 nm, and the concentration was determined by comparing the optical density value of samples with the standard curve. The level of total collagens was calculated as μg/ml of protein and reported as fold change compared to the vehicle group.

Immunohistochemistry

For immunohistochemistry, the xenografts were fixed with 4% paraformaldehyde in PBS (phosphate-buffered saline) and subsequently transferred to PBS containing 30% sucrose (wt/vol) until equilibrated in cold (4 °C). After fixation, 5-μm-thick paraffin sections were treated three times with HistoClear^™ (National Diagnostics, Atlanta, GA, USA) for 5 min, rehydrated by a sequential ethanol wash, and then incubated in a target-retrieval solution (Dako, Carpinteria, CA, USA) in a microwave for 10 min in order to retrieve the antigens. For blocking, tissues were incubated for 10 min with a 3% solution of H₂O₂ followed by incubation with PBS-5% normal goat serum-0.2% Triton X–100. Tissue sections were incubated with primary antibody rabbit anti-Ki67 (dilution 1:250, 27309-1-AP, Proteintech Group, Inc, Rosemont, IL, USA), rabbit anti-cleaved caspase-3 (dilution 1:50, #9664, Cell Signaling Technology, Danvers, MA, USA), and mouse anti-E2F1 (dilution 1:50, sc-251, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4 °C in a humidified chamber. The antigens were then visualized using biotinylated antibodies and streptavidin, conjugated with horseradish peroxidase. Control sections were incubated with the secondary antibody, with replacement of the primary antibody with the dilution reagent (Dako). Diaminobenzidine (Dako) served as the substrate. All the sections were counterstained with hematoxylin and eosin. Immunostained sections were examined under a microscope (Olympus IX83; Olympus Surgical Technologies America, San Jose, CA, USA), and five representative images of each slide were quantitatively analyzed with the use of Halo software (Indica Labs Inc., Albuquerque, NM, USA) and were averaged for comparative analysis between vehicle and 680C91 treatment groups in a blinded fashion.

Fibroid Explant Culture

Equal weights of fibroid explants (4–5-mm³ cubes) cut aseptically from the same patient were plated in 6-well in complete medium and incubated for 48 hours with tryptophan (0.8 mM) or tryptophan plus AhR antagonist (CH-223191; 10μM) or TDO2 inhibitor (680C91; 50μM).

Statistics and Power Analysis

Throughout the text, results are presented as mean ± SEM and data was analyzed by GraphPad PRISM 10 software (Graph-Pad, San Diego, CA, USA). Dataset normality was determined by the Kolmogorov–Smirnoff test. The data presented in this study was not normally distributed, and therefore nonparametric tests were used for data analysis. Comparisons involving two groups was analyzed using the Wilcoxon matched pairs signed rank test as appropriate. The Kruskal-Wallis Test was used for comparisons involving multiple groups (Fig. 4D). The relationship between two expression groups was determined using Spearman r correlation coefficient. Statistical significance was established at p < 0.05.

Figure. 4.

Correlation analysis between the fold change in (A) tumor weight with kynurenine level in the xenografts; (B) xenograft kynurenine level with FN1 protein; and (C) tumor weight with FN1 mRNA level in the xenografts (D) Fibroid explants (N=3) were treated with tryptophan (0.8 mM) or tryptophan plus AhR antagonist (CH-223191; 10μM) or TDO2 inhibitor (680C91; 50μM) for 48 hours and mRNA expression was determined by real time PCR. The results are presented as mean ± SEM with p values indicated by corresponding lines. *p < 0.05; **p < 0.01; ***p < 0.001.

Results

The administration of 680C91 was well-tolerated. Supplementary Table 1 shows the results of blood chemistry after two months of treatment with the drug. There were no effects of 690C91 on liver, kidney or pancreatic function and no effects on body weight of the animals.

There was about a 30% reduction in the weight of fibroid explants in mice treated with 680C91 (Fig. 1A). Representative images of fibroid xenografts treated with vehicle or 680C91 are shown in Fig. 1B. As expected, there was a reduction in kynurenine levels (Fig. 1C) in the xenografts from animals treated with 680C91 as compared with vehicle treated mice which indicates that the drug effectively inhibited TDO2 enzyme activity. The xenografts were analyzed for a number of key genes known to play a role in fibroid pathogenesis. As shown in Fig. 2A treatment with the inhibitor resulted in decreased mRNA expression of the pro-fibrotic cytokine TGF-β3 (17–19), the inflammatory gene marker IL-8 (20), the cell cycle regulatory genes (E2F1 and CDK2) (17, 21), FN1, a major component of the ECM (22) and SPARC, a matricellular protein involved in ECM remodeling and composition (23). Treatment with 680C91 also inhibited the expression of CYP1B1, a gene regulated by AhR and a marker for its activation (24). We also measured total collagen protein levels in the xenografts and as shown in Fig. 2B there was a significant decrease in total collagen protein levels in xenografts of mice treated with 680C91. The levels of CYP1B1, FN1 and SPARC were also measured by Western blot analysis and in concordance with the mRNA expression there was decreased protein abundance in the xenografts of mice treated with 680C91 as compared with vehicle treated mice (Figs. 2C–D). We did not find significant effect of the inhibitor on TGF-β3, IL-8 and CDK2 at the protein levels as determined by ELISA and Western blot analysis (data not shown).

Figure 1.

(A) Fresh fibroid explants were implanted subcutaneously in the flank of ovariectomized CB-17 SCID/Beige mice and treated i.p daily for two months with the vehicle or TDO2 inhibitor (680C91; 10 mg/Kg). The weight of tumor explants was determined after 8 weeks of treatment (n=12). (B) Representative images of four xenografts at the end of treatment period (8 weeks). (C) Kynurenine levels as determined by enzyme-linked immunosorbent assay in 12 xenografts. The results are presented as mean ± SEM of independent experiments with p values indicated at the corresponding line. *p <0.05.

Figure 2.

(A) Relative mRNA expression of FN1, CYP1B1, E2F1, CDK2, TGF-β3, IL8 and SPARC in (A) xenografts implanted subcutaneously in the ovariectomized CB-17 SCID/Beige mice (n=12) following 8 weeks of treatment with vehicle or TDO2 inhibitor (680C91; 10 mg/Kg). (B) Total collagen levels as determined by enzyme-linked immunosorbent assay in 12 xenografts. (C) Representative Western blot analysis of CYP1B1, FN1 and SPARC with bar graphs (D) showing their relative band densities in the xenografts (n=12). The results are presented as mean ± SEM of independent experiments with p values indicated at the corresponding line. *p < 0.05; **p < 0.01; ***p < 0.001.

The xenografts were also subjected to histologic and immunohistochemical staining and image analysis using Halo software (Fig. 3). As shown in the top panel (Figs. 3A–B) staining of xenograft section with Masson’s trichrome stain showed that explants from the 680C91-treated mice had significantly reduced staining for collagen as indicated by the blue stain. These xenografts also showed a significant decrease in cell proliferation as indicated by the reduced nuclear staining for Ki67 (Figs. 3C–D), but no significant change in apoptosis as indicated by cleaved caspase 3 staining (Figs. 3E–F). There was also a significant reduction in staining for E2F1, a cell cycle regulatory protein (Figs. 3G–H) consistent with the mRNA expression.

Figure 3.

(A-B) Representative histopathological images determined by Masson’s trichrome staining of fibroid xenografts from vehicle or TDO2 inhibitor-treated group. Blue color demonstrates collagen fibers and red color indicates smooth muscle cells . (B) Shows the quantification of staining intensity by Halo software. (C-H) Representative immuno-histochemical stained images of fibroid xenografts treated with vehicle or TDO2 inhibitor for Ki67 (C-D), Cleaved Caspase 3 (E-F) and E2F1 (G-H). Image analysis of nuclear staining was done by Halo software. The results are presented as mean ± SEM with p values indicated at the corresponding line. *p <0.05.

We performed correlation analysis between the levels of kynurenine in the xenografts and various parameters including tumor weight (Fig. 1A) and FN1 levels (Fig. 2). As shown in Figs. 4 A and 4B there is a significant direct correlation between the xenograft kynurenine levels and tumor weight and FN1 protein. We also detected a significant correlation between the tumor weight and FN1 mRNA (Fig. 4C).

In order to determine the mechanism by which the TDO2 inhibitor exerts its effects in vitro studies were carried out with fibroid explants treated with tryptophan alone and tryptophan plus the 680C91 and the AhR antagonist CH-223191. After 48 hours of culture the explants were analyzed for the expression of the same genes (Fig. 2) we found to be significantly altered by in vivo administration of 680C91. As shown in Fig. 4D tryptophan significantly induced the expression of TGF-β3, SPARC, IL-8, E2F1, CDK2, FN1 and CYP1B1 mRNA. The effects of tryptophan were blocked by co-treatment with the TDO2 inhibitor 680C91 and by the AhR antagonist CH-223191 indicating that the observed in vivo effects of 680C91 are mediated by AhR pathway.

Discussion

The results of this preclinical study indicate that treatment of mice bearing human fibroid xenografts with compound 680C91, a specific inhibitor of TDO2 results in 30% reduction in tumor weight after two months of treatment. In response to the inhibitor treatment there was a significant reduction in kynurenine levels in the xenografts indicating efficient inhibition of TDO2 to break down tryptophan to kynurenine. Furthermore, we found a direct correlation between kynurenine levels in the xenografts and the decrease in fibroid weight and the expression of FN1, a major component of the ECM. Gene profiling of the xenografts from the compound 680C91 treated mice as compared with matched xenografts from vehicle treated mice revealed a significant reduction in TGF-β3, FN1, CDK2, E2F1, IL-8, SPARC, and CYP1B1 mRNA expression. These xenografts also expressed lower total collagen, FN1 and CYP1B1 protein levels as compared with vehicle treated animals. Immunohistochemical and histologic analysis of xenografts indicated a decreased expression of collagen, Ki67, E2F1 in mice treated with the inhibitor. In vitro studies with fibroid explants treated with tryptophan indicated an induction of TGF-β3, FN1, CDK2, E2F1, IL-8, SPARC and CYP1B1 mRNA and this induction could be blocked by both 680C91 and the AhR antagonist CH-223191 indicating that the observed in vivo effects of 680C91 are mediated by the AhR pathway. The promising results from this preclinical in vivo study with the inhibitor of TDO2 indicate that correction of markedly dysregulated degradation of tryptophan previously demonstrated by our group (4, 5) could be an effective therapy for fibroids.

The degradation of tryptophan by TDO2 results in production of kynurenine, which is a ligand for AhR, a transcription factor and member of the basic helix–loop–helix/Per-ARNT-SIM (bHLH-PAS) family (25). Our results indicate that inhibition of TDO2 results in downregulation of CYP1B1 which is a downstream gene regulated by AhR. After binding of AhR to its ligand it dimerizes with AhR nuclear translocator (ARNT) and acts as a transcription factor affecting transcription of wide array of genes that bear the xenobiotic response element (25). AhR signaling is involved in diverse processes many of which are relevant to fibroid pathophysiology. AhR signaling stimulates oxidative stress (26), inhibits autophagy (27–29) and is involved in the inflammatory process though its interaction with NF-κB (30, 31). This signaling pathway is also involved in the differentiation of immune cells by enhancing the generation of immunosuppressive phenotype (31).

Our data indicate that the decrease in the weight of fibroid explants in response to TDO2 inhibitor treatment is primarily due to the drug effect on cell proliferation and not cellular apoptosis. This decrease in cell proliferation is evidenced by decreased Ki67 expression in xenograft from animals treated with 680C91, and decreased expression of genes critical in cell cycle regulation namely CDK2, a serine/threonine protein kinase which regulates G1/S phase transition (32), and E2F1 a transcription factor which plays a critical role in the control of cell cycle and apoptosis (33). Both E2F1 and CDK2 are overexpressed in fibroids (17, 21). E2F1 also directly interacts with AhR displacing p300 protein from the complex resulting in the inhibition of transcription of many E2F regulated genes which control the S phase progression (34). The interaction between E2F and AhR was shown to also lead to inhibition of E2F1-induced apoptosis in mouse hepatoma cells and human osteosarcoma cells (35). In addition to its inhibitory effects on cell proliferation, the reduction in fibroid xenografts weight following the TDO2 inhibitor treatment could be secondary to a reduction in ECM volume as evidenced by decreased expression of collagens and FN1, and the direct correlation between FN1 levels, a major component of the ECM and tumor weight.

A hallmark of fibroids is the accumulation of ECM with overexpression of collagen and fibronectin (1–3). Additionally, these tumors overexpress TGF-β3 which is a master regulator of fibrosis (17–19). Compound 680C91 inhibited the expression of TGF-β3 mRNA and the expression of collagens and FN1 protein in the fibroid xenografts. Some of these effects could be secondary to the inhibition of AhR signaling as evidenced by decreased CYP1B1 levels in the xenografts of the inhibitor treated mice, and our in vitro data showing blockade of tryptophan induction of TGF-β3 and FN1 by the AhR antagonist. The TDO2 inhibitor also inhibited the expression of SPARC in vivo, and our in vitro finding indicated that this effect is mediated by AhR. Recently our group reported a marked overexpression of SPARC in fibroids as compared with their matched myometrium (36). SPARC is a matricellular secreted glycoprotein which directly binds to the ECM collagen and activates MMPs (37, 38) and indirectly regulates growth factors involved in angiogenesis and tissue remodeling including FGF, VEGF and TGF-β (23). AhR signaling is known to interact with the NF-κB pathway (30, 31, 39) and TGF signaling pathway (40) both of pathways playing critical roles in fibroid pathogenesis (1–3).

Another mechanism by which 680C91 can exert its effects could be immune mediated. AhR activation by kynurenine dampens the immune response to prevent excessive inflammation and autoimmunity by production of excess amounts of immunosuppressive metabolites of the kynurenine pathway (41). The metabolites of the kynurenine degradation pathway have immunosuppressive role due to their ability to suppress T cell reactivity and represents a mechanism by which tumors escape an immune response. Multiple clinical trials are underway to examine the efficacy of TDO2/IDO1 inhibitors with the goal to reduce kynurenine and other immunosuppressive tryptophan catabolites, thereby reverting the immunosuppressive microenvironment induced by tryptophan catabolites. These drugs which resemble tryptophan (also referred to as checkpoint inhibitors) have shown promise for cancer immunotherapy (42). Because these drugs mimic tryptophan they can have off target effects and can activate the AhR. The activation of AhR could have pro-carcinogenic effects in some human cancers like lung, prostate, breast, and pancreatic cancer whereas in other cancers like colon cancer it could have anti-carcinogenic effect (43). In this study 680C91 did not have an activation effect on AhR. The activation of AhR by kynurenine also has epigenetic effects that could lead to chromatin remodeling. The AhR-ARNT interacts with several histone acetyltransferases and chromatin remodeling factors to block histone acetylation (44).

In summary, our data indicate that inhibition of TDO2 expression by 680C91 has beneficial effects by reducing fibroid growth through its inhibitory effect on cell proliferation. In addition, the inhibitor treatment induced a favorable gene profile in the fibroid with a reduction in the ECM volume as indicated by decreased collagens and FN1 expression, reduced inflammation as indicated by decreased expression of inflammatory genes, SPARC and IL-8 and decreased fibrosis as indicated by reduced TGF-β3 expression in the tumors. The effects of compound 680C91 are most likely due to its effect to reduce kynurenine production and thereby decreased AhR activation as evidenced by our findings of lower CYP1B1 mRNA/protein in fibroid xenografts and our in vitro experiments with fibroid explants. These promising therapeutic effects of TDO2 inhibitor for the treatment of fibroids warrant additional animal studies and phase 1 human clinical trial.