1'H-Indole-3'-Carbonyl-Thiazole-4-Carboxylic Acid Methyl Ester Blocked Human Glioma Cell Invasion via Aryl Hydrocarbon Receptor's Regulation of Cytoskeletal Contraction.

Blocking glioma cell invasion has been challenging due to cancer cells that can swiftly switch their migration mode, and agents that can block more than one migration mode are sought after. We found that small molecule 2-(1H-indole-3-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), an endogenous aryl hydrocarbon receptor (AHR) agonist, can block more than one mode of glioma cell migration, based on cultured cell behavior captured by videos. Data from wound-healing assays and mouse xenograft glioma models corroborated ITE's migration-inhibiting effects while knocking down AHR by siRNA abolished these effects. To identify genes that mediated ITE-AHR's effect, we first collected gene expression changes upon ITE treatment by RNA-seq, then compared them against literature reported migration-related genes in glioma and that were potentially regulated by AHR. MYH9, a component of nonmuscle myosin IIA (NMIIA), was confirmed to be reduced by ITE treatment. When MYH9 was overexpressed in the glioma cells, a good correlation was observed between the expression level and the cell migration ability, determined by wound-healing assay. Correspondingly, overexpression of MYH9 abrogated ITE's migration-inhibiting effects, indicating that ITE-AHR inhibited cell migration via inhibiting MYH9 expression. MYH9 is essential for cell migration in 3D confined space and not a discovered target of AHR; the fact that ITE affects MYH9 via AHR opens a new research and development avenue.

1. Introduction

Glioblastoma (GBM), the most common and advanced form of glioma, is one of the most lethal human cancers. GBM patients survive, on average, 12–15 months despite aggressive surgical resection, radiation, and chemotherapy [1]. The diffuse invasion of the GBM tumor cells and the blood-brain barrier pose major challenges for successful therapy since invaded tumor cells are sheltered from complete surgical removal and radiation. Targeted drug discovery concentrates on the receptor and signaling pathway, such as EGFR, VEGFR, PDGFR, MET, MEK, and molecules that mediate cell interaction with the ECM, such as integrin and syndecan.

We targeted AHR, a ligand-activated transcripn>tion factor, and E3 ubiquitin ligase. pan class="Gene">AHR regulates cytotoxic immune cells in cancer by controlling the function and differentiation of multiple types of immune cells. In glioma, AHR is expressed in glioma and the stromal cells, including endothelial cells, astrocytes, oligodendrocytes, microglia, macrophages, dendritic cells, and T lymphocyte [2-4]. AHR's effects on cancer are complex, highly depends on the ligands in the microenvironment and the cell types. Glioma, its reported that AHR regulated tumor cell migration and may serve as a tumor suppressor [5].

We found that targeting AHR in pan class="Disease">glioma blocked glioma invasion. The majority of endogenous AHR ligands are tryptophan metabolites, and the most abundant tryptophan metabolite in glioma is kynurenine, which is produced by enzymes 2,3-dioxygenase (IDO/TDO), binds to AHR and inhibits cytotoxic immune cell activity against glioma cells [6], forming a pathway of Tryptophan-Kynurenine-AHR-Impairment of Immunity. To block this pathway, inhibitors for the IDO/TDO were developed and mixed clinical trial results have been posted [7, 8], not meeting expectations. An alternative route is to directly target AHR using small-molecule ligands. We hypothesized that targeting AHR using a high-affinity agonist could achieve this goal while maintaining AHR's tumor suppressor effects in glioma. To this end, we tested the effects of ITE, an endogenous AHR agonist, and discovered that ITE significantly inhibited glioma cell migration and invasion.

We report that ITE can significantly block glioma cell migration and invasion in wound healing and transwell assay, in brain invasion assay, and in a mouse model. In a 3D collagen matrix, in addition to the more dynamic filopodia, ITE induced weaker protrusion attachment to the ECM. In 2D cultures, glioma cells failed to undergo amoeboid migration when treated with ITE and subject to pH changes. ITE blocked the invasion of the tumor into the surrounding brain parenchyma.

2. Materials and Methods

2.1. Cell Culture

Human and mouse glioma cell lines U87MG and GL261 were obtained from the National Infrastructure of Cell Line Resource and the Third Military Medical University, respectively. U87MG and GL261 cells were cultured using MEM/EBSS and DMEM/F12 supplemented with 10%FBS, respectively.

2.2. Cell Invasion and Migration Assays

Boyden chamber (CORNING, USA) was coated with 30 μl rat tail tendon collagen type I (PERFEMIKER, #C20-200110), incubated for 30 min at 37°C. 5 × 104 cells were seeded into chambers and cultured in DMEM/F12 with 2% FBS, and the lower chamber medium contains 10% FBS. After 20 hr, cells were fixed with 4% paraformaldehyde, and the nonmigrated cells were removed with cotton swabs. The migrated cells on the bottom were stained with crystal violet and counted. Invasion assays were repeated six times over multiple days.

For the wound-healing assay, horizontal lines were drawn on the back of 12-well plates at a spacing of 0.5~1 cm using a markpan class="Gene">er pen, with at least 5 lines pass through each well plate. When cells were grown to 70% confluence in 12 well tissue culture dish, a single scratch was made perpendicular to the horizontal lines, using a 10 μl pipette tip. Cells were then rinsed with PBS and incubated in serum-free medium with different concentrations of ITE (0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM, and 100000 nM) or 0.02% DMSO (SIGMA, #D5879) for 20 hr. Images were captured at 0 hr and 20 hr at the same position of cross points of horizontal lines and scratch. Using ImageJ, the wound in each image was identified using the magic wand tool, and the area measured by the ROI function. Wound-healing assays were carried out in three batches of the experiment. In a batch, each treatment was replicated in three wells, quantified by measuring two or three fields per well.

2.3. Mouse Orthotopic Glioma Model

All experiments were performed following the animal care guidelines of Inner Mongolia Agricultural University.

8-week-old C57BL/6 mice (Beijing Vital Rivpan class="Gene">er Laboratory Animal Technology Co. Ltd) were anesthetized with an intraperitoneal injection of 0.3% pentobarbital sodium (30 mg/kg). For the stereotactic intracranial injection, the surgical site was shaved and prepared with iodine. A midline incision was made to expose the bregma point, and a 1 mm diameter right parietal burr hole was drilled with a dental drill, centered at 2 mm posterior to the coronal suture and 2 mm lateral to the sagittal suture. Mice were placed in a stereotactic frame and 1 × 105 GL261 cells in 2.5 μl medium were intracranially injected at a depth of 3 mm using Microinjection Pump (KD scientific, 78-1311Q) at a speed of 0.5 μl/min. The needle was removed slowly, and the skin was sutured with nylon thread.

2.4. Hematoxylin and Eosin Stain of the Mouse Brain Tissue

Mice were anesthetized and perfused with 4% paraformaldehyde (PFA) through the right atrium. Brains were harvested and placed in 4% PFA for 12 hr, then in 15%, 20%, and 30% sucrose solutions consequently for 12 hr each. Tissue was embedded in Optimal Tissue Cutting Compound (OCT# SAKURA) and frozen in liquid nitrogen. Tissue was sectioned at 12 μM using frozen slicer (LEICA #CM1850 Germany). Sections were then stained with hematoxylin and eosin (Sigma Aldrich, #SLBS9620) and imaged using microscope and stereomicroscope (Nikon, Tokyo, Japan).

2.5. Measurement of Tumor Invasion in the Mouse Model

The extent of tumor invasion in the mouse model was measured in H&E staining images of mouse brain frozen sections, using convexity by the following equation:

The tumor edge on the images was found using ImageJ: (1) Primary edge finding was done by manually draw along the tumor edge using the magic wand tool, added to ROI, and apply the find edge function. (2) Precise edge finding was done by first adjust the image threshold, then apply the ABS snake plugin utilizing the primary edge found in the 1st step.

Tumor edge was saved as a mask and applied to the original image, and the magic wand tool was used for perimeter measurement. The convex perimeter was measured using the convex hull tool. Three animals from each treatment were subjected to this invasion analysis, and the t-test was applied to compare the convexity of the ITE and DMSO group.

2.6. Organotypic Brain Slice Culture

Mouse brains wpan class="Gene">ere extracted from 6-week-old female C57BL/6 mice (euthanized by cervical dislocation) and placed in PBS solution with 3% penicillin and streptomycin in a sterile petri dish. Three percent low melting point agarose (Solarbio, #A8350) solution was prepared and cooled to touch, then poured over the brain, and cooled for an additional 30-45 min. The agarose gel was trimmed, and 300 μM organotypic brain slices were generated using a vibrating microtome ZQP-86 (Zhi Xin Biotech, Shang Hai, China). The brain slices were sectioned into two symmetrical halves and transferred to 6-well transwell plate chamber by spatula and placed next to each other. Two milliliters of DMEM/F12 medium with 25% FBS was added to the lower chamber and placed in a 37°C and 5% CO2 incubator for overnight culture.

2.7. Organotypic Brain Slice Invasion Assay

105 GL261 cells wpan class="Gene">ere incubated with CM-Dil (YEASEN, # 40718ES60) for 15 min in 2 μl media, seeded onto the seam where two brain slices met, and the brain slices were cultured at the air-liquid interface, with the culture media refreshed every 2-3 days. For drug treatments, serum-free media were added to the lower chamber, with different doses of ITE (0.1 nM, 10 nM, 1000 nM, and 3000 nM) FAK inhibitor PF431396 (MCE, # 717906-29-1) or 0.02% DMSO control. After 5 days and 8 days of continuous culture, cell invasion was observed under a stereomicroscope (Nikon, Tokyo, Japan) for overall slice imaging. For high-resolution imaging, multiple overlapping pictures were taken and stitched together using the software panoramamaker6.

2.8. 3D Culture of GL261 Cells

2 × 105 cells at 50 μl were mixed with 12 μl pan class="Chemical">NaOH, 710 μl complete medium, and 23 μl PBS, as well as 220 μl rat tail tendon collagen type I (PERFEMIKER, #C20-200110). After solidification, supplement 1 ml complete medium was added to the gel. Time-lapse photography was performed using an OlympusIx71 microscope with software Cell Sens Standard, at 30 s intervals for 1 hour. Time-lapse videos were made using ImageJ.

2.9. AHR siRNA Gene Knockdown and Total RNA Extraction

8 × 105 U87MG cells were seeded into a 6-well plate and transfected when cells grow to 60% confluence according to the manufacturer's instructions. After 4 hr of transfection, fresh complete medium was added, and total RNA was extracted from the cells after 36 hr transfection using RNA Fastagen Kit (Shanghai Feijie Bio, #220010) according to the manufacturer's protocol.

2.10. Western Blot

Protein was extracted on ice in RIPA solution (Solarbio, #R0020) and quantified using a pan class="Chemical">bicinchoninic acid protein assay kit (Solarbio, #PC0020). Forty-microgram protein was separated by 8% SDS/PAGE and transferred to the nitrocellulose membranes (MILLIPORE, #HATF00010) at 120 v for 90 min. Membranes were blocked with 5% Skim Milk solution (Solarbio, #D8340) for 3 h at 37°C, immunoblotted with primary antibody (listed in Supplementary Table 1) overnight at 4°C, washed, then with a secondary antibody, washed, and imaged using ODYSSEY CLX (Clx-0519, LI-COR, Gene Company Limited, USA).

2.11. Transient Transfection of Cells

8 × 105 U87MG cells were plated into 6-well plates 24 hr before transfection, and the cells were washed and supplemented with 2 ml DMEM before transfection. Plasmid DNA and PEI (ALDRICH, Lot #BCBT0649) were each diluted with MEM/EBSS and allowed to stand for 10 min. The DNA solution was added into the PEI solution and mixed well at N/P = 20, incubated for 10 min. PEI/DNA complexes were added to the cells and replaced by fresh medium after 3.5 hr. The empty vector was employed as a control. MYH9 transgene expression was verified using real-time PCR.

2.12. Real-Time PCR

Four hundred nanograms of total RNA was employed for cDNA synthesis using ProtoScripn>t II First Strand cDNA Synthesis pan class="Gene">kit (TaKaRa, #RR036A) according to the manufacturer's protocol. Forty nanograms of cDNA was used as templates for RT-PCR using the SYBR® Premix Ex Taq kit (TaKaRa, #RR820A) using Light Cycler W 480 (Roche, Switzerland). The sequences and efficiency of all primers were listed in (Supplementary Table 2), and HPRT/GAPDH was used as reference genes.

2.13. Statistical Analysis

All data were presented as the mean ± standard deviation. One-way ANOVA was utilized to determine the statistical significance of the differences in GL261 and U87MG cell migration, invasion, and western blot analysis. A repeated-measure ANOVA was used to analyze how AHR siRNA and different ITE doses affect cell migration. Data were expressed as means ± SD. The results are displayed as the mean ± standard deviation for n ≥ 3 samples per group in all cases, and P < 0.05 was considered significant differences.

3. Results

3.1. ITE Inhibited Glioma Migration and Invasion In Vitro, Ex Vivo, and In Vivo

We investigated ITE's effects on glioma cells using wound-healing migration and transwell invasion assays. 100, 1000, and 10000 nM ITE significantly inhibited U87MG and GL261 migration (Figures 1(a) and 1(b)). We further tested ITE's effects on cell invasion using the Boyden chamber assay and applied a known invasion blocking small-molecule new drug candidate, and FAK inhibitor PF431396 as a positive control. 10 nM ITE significantly inhibited both U87MG and GL261 cell invasion (Figures 1(c) and 1(d)).

Figure 1

ITE inhibited migration and invasion of glioma cells in wound healing and the Boyden chamber invasion assay. (a, b) Wound-healing assays. U87 and GL261 cells were treated with DSMO or various doses of ITE for 20 hr. The area of the wound was measured in 3 replicate wells per experiment (∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001), with a drug treatment experiment replicated 3 times. (c, d) Boyden chamber invasion assays. The cells were treated with various concentrations of ITE, DMSO, or PF431396 (FAK inhibitor) for 20 hr. Those migrated through the rat tail tendon collagen type I gel and the membrane were stained and counted (∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001). (e–f) siRNA knocking down of AHR expression in U87MG cells. AHR siRNAs or nontargeting siRNA control (NC) were transfected into the U87MG cells for 36 h, and the AHR level was assessed by RT-PCR and western blot. (g) For drug treatment, U87MG cells were transfected with AHR siRNA or with NC for 36 hr and treated with DMSO or various concentrations of ITE for additional 20 hr. Cell migration was assessed by wound-healing assays, and the data from three batches of drug treatment experiments were analyzed using repeated-measure ANOVA (∗ statistical significance for difference within group and # statistical significance for difference between groups; ∗ or #P < 0.05, ∗∗ or ##P < 0.01, and ∗∗∗ or ###P < 0.001).

We knocked down AHR expression in U87MG cells by siRNA (Figures 1(e)–1(f)) and found that 100, 1000, and 10000 nM ITE significantly inhibited cell migration in the nontargeting control (NC) siRNA group, showing similar efficacy as in untransfected cells. In the AHR siRNA knockdown group, migration was significantly reduced in the DMSO, 0.1, 1, and 10 nM ITE groups, highlighting AHR's role in regulating glioma cell migration. In AHR knockdown cells, higher ITE doses (100, 1000, and 10000 nM) failed to further inhibit cell migration in siRNA-transfected cells, indicating that ITE's effect depended on sufficient AHR expression level (Figure 1(g)).

To test whether ITE can block invasion into the brain parenchyma, we performed ex vivo brain invasion assay using live-cell dye CM-Dil labeled GL261 cells (Figures 2(a) and 2(b)). We found that ITE significantly inhibited GL261's invasion into the normal mouse brain parenchyma at 10 nM, showing a high efficacy compared to the positive control drug PF431396, corroborating that ITE was a potent inhibitor for glioma cell invasion.

Figure 2

ITE blocked glioma cell invasion into the brain parenchyma in an ex vivo brain slice model and an orthotopic mouse model of glioma. (a) Whole-brain images of labeled GL261 cells invade into cultured mouse brain slices. (b) Cells were incubated with CM-Dil Dye, seeded into the seam of two adjacent brain slice halves cultured at the air-liquid surface with DMSO, a various dosage of ITE, or FAK inhibitor PF431396. Representative whole-brain slice images were obtained on day1, day5, and day8. High-resolution brain slice culture images obtained by stitching overlapping images. (c) Experimental design and treatment schema. (d) Glioma invasion observed in the orthotopic mouse model. (I) Gross appearance of a mouse brain with a glioma in the cerebrum. (II) Representative images of whole-brain horizontal frozen sections with H&E staining showing glioma invasion toward the ventricles. (III) Representative image showing glioma invasion into the brain parenchyma and toward the blood vessel. Arrows, ventricles. Asterisk, invading tumor cells at the tumor edge. Star, blood vessel. (e) Local invasion patterns at tumor edges of the ITE or DMSO group. Tumor-bearing mice were treated with DMSO or 100 mg/kg body weight ITE and were euthanized. Horizontal brain sections from two animals per group were shown. (f) Convexity of the tumor shape in brain tissue sections as a measure of tumor invasion. The convexity of the tumor was done by first finding the edge of the tumor using ImageJ, then measure the perimeter and convex perimeter of the tumor shape, and t-test was applied for data analysis.

We tested whether pan class="Chemical">ITE can block invasion in an orthotopic mouse model of glioma using GL261 cells injected into the front lobe of the mouse brain (Figure 2(c)). Typical glioma invasion patterns were observed in this model, including into the peritumor parenchyma, toward the ventricles or toward the blood vessels (Figure 2(d)). Locally at tumor edges, ITE-treated tumors shown more smooth edge hence less invasion into the parenchyma (Figure 2(e)). For overall invasion assessment, we adopted the convexity, a mathematical measurement of shape curvature, calculated as a ratio between convex perimeter and perimeter of overall tumor shape in the H&E-stained tissue sections. ITE (100 mg/kg body weight, given every other day) significantly increased the convexity of the tumor shape. (Figure 2(f)).

3.2. ITE Inhibited Polarity, Protrusion Attachment and Rear Contraction of 3D Cultured Cells

As the first step of the mechanistic study, we plated glioma cells in the type I collagen matrix, treated them with pan class="Chemical">ITE or DMSO for 20 hr, the same duration we adopted in the in vitro migration/invasion assays. While the DMSO cells were polarized, only spherical nonmotile cells that continuously bleb were observed (Video. 3A-B). To rule out the possibility of apoptosis, we performed a CCK experiment using the same dose and duration (Supplemental Figure 1) and continuously photographed these spherical cells for 3 hr. No sign of apoptosis was observed in either the CCK experiment or the cell behavior under a microscope. Sign of cellular polarization such as a stable large bleb on one side or multiple dynamic blebs on all but one side of the cells was not seen in these cells, indicating that ITE treatment prevented cell polarization.

Shorter (1 hr) ITE treatment provoked cytoskeletal changes in U87MG cells. The more spreading cell body and frequent filopodia extension and retraction were observed (Video. 3C-D). In the control group, when a prominent lamellipodium-like protrusion detached, a shake of the extracellular matrix was observed (Video. 3C), indicating a strong attachment, which was not found in the ITE-treated group (Video. 3D). Furthermore, the prevalent protrusion of ITE-treated cells has more vibrant ruffles at the tip, confirming an unstable attachment to the ECM.

Efficient mesenchymal migration was observed in the control group cells, overcoming constrictions in the matrix (Video. 3E). However, in the ITE-treated cells, the cell rear remains attached to the ECM while its front part moves forward, resulted in cells with an elongated rear (Video. 3F), indicating compromised cellular contraction.

3.3. ITE Inhibited Switching to the Amoeboid Migration Modes

In the DMSO 1 hr treatment group, we observed mesenchymal migrating cells started to bleb when encountering matrix constrictions (Video. 3E). Moreover, the A1 mode of the amoeboid migration, characterized by a prominent leading protrusion and round cell shape, was also observed [9] (Video. 3A), indicating that cells could swiftly switch to amoeboid migration when encountering space restrictions. No such phenomenon was observed in the ITE-treated group, and typical cells still adopted the elongated shape (Video. 3F).

Accordingly, ITE-treated cells in 2D culture also failed to undergo amoeboid migration when subject to pH changes. When taken out of the incubator for 2 hr photography, the culture medium became basic and U87MG cells responded to the pH change by becoming spherical and migrating, which is evident in the overlapping photos taken at the beginning and end of the photography period (Figure 3(g), Video. 3I). ITE-treated cells remained in the same position and maintained the same shape failed to respond (Figure 3(h), Video. 3J). The few spherical cells in the ITE-treated group also lack the sign of polarity.

Figure 3

Effects of ITE on the spreading, protrusion attachment, and migration of glioma cells in 2D and 3D collagen matrix. (a–f) U87MG cells were plated in the type I collagen matrix, settled overnight, treated with either ITE or DMSO, and recorded by time-lapse photography: (a, b) Cells were treated with DMSO or ITE for 20 hr. (c, d) Cells were treated with DMSO or ITE for 1 hr. (e–f) Cells were treated with DMSO or ITE for 2 hr. (g–j) U87MG cells in 2D culture were treated with DMSO or ITE for 18 hr and recorded for 2 hr. (g, h) Overlap of photos taken at the beginning (cell edge colored in red) and end of the 2 hr (cell edge colored in green) using ImageJ. (i, j) Videos of U87MG cells in 2D culture.

3.4. MYH9 Mediated ITE Migration-Inhibiting Effects in Human Glioma Cells

Since ITE-treated cells display cytoskeletal changes, we studied related molecules. Sevpan class="Gene">eral integrin, collagen, and FAK were examined by either RT-PCR or western blot, and none showed significant changes (Supplemental Figure. 2A-D). To identify the genes that mediated ITE's invasion-inhibition effects, we compared gene expression profiles of ITE versus DMSO-treated U87MG and GL261 cells. Candidate genes were filtered by comparing the differently expressed genes from our experiment, a reported glioma migration-related gene set, and reported AHR-regulated genes. The genes in common contained mainly TGF beta pathway members and MYH9 (Supplemental Figure. 3A, Table 3). No significant changes in Smad2/3 protein were detected when comparing ITE to DMSO-treated U87MG cells (Supplemental Figure. 3B), and MYH9 was further studied.

MYH9, a component of the nonmuscle myosin IIA (NMIIA), was significantly reduced by various doses of ITE treatment (0.1, 1, 10, 100, and 1000 nM) in U87MG cells, both by RT-PCR and western blot analysis (Figures 4(a) and 4(b)). To confirm the relationship between ITE and MYH9, we transfected into the cells with MYH9 cDNA fused to mCherry. Imaging, RT-PCR, and western blot confirmed elevated MYH9 levels in transfected cells (Figures 4(c)–4(e)). Again, various doses of ITE (0.1, 10, and 1000 nM) significantly reduced MYH9 mRNA expression in both groups (Figure 4(f)), indicating that ITE inhibited MYH9 expression across a wide background expression range.

Figure 4

MYH9 expression level and cell migration ability. (a) Cells were treated with ITE for 18 hr, and mRNA levels of MYH9 were determined by RT-PCR using GAPDH as a reference gene. (b) MYH9 protein levels were assessed in U87MG by western blots. The bar graph summarizes the semiquantitation of the western blot data. (c–e) Overexpression of MYH9-mCherry fusion protein by transfection of the U87MG cells. (c) MYH9 gene overexpressed in U87MG cell after 36 h transfection; the left panel is the bright field, the middle panel is red fluorescence from mCherry, and the right panel is the merged picture. (d, e) MYH9 mRNA and protein level were determined by RT-PCR and western blot using GAPDH as a reference gene (∗∗P < 0.01) in MYH9 transfected an empty vector (PCMV) or untransfected cells. (f) ITE reduced MYH9 mRNA level in U87MG cells with normal or elevated MYH9 level. MYH9 or empty vector-transfected cells were treated with either ITE or DMSO for 18 hr, and MYH9 mRNA levels were determined. (g) ITE's effect on the migration ability of the U87MG cells that overexpressing MYH9. Either MYH9 plasmid or empty vector-transfected cells were treated with various doses of ITE or DMSO for 20 hr. The whole experiments were replicated 3 times, and the area of migration was measured using 2 replicate wells in each batch. ∗: various ITE concentrations compared with DMSO treatment in vector; #: various ITE concentration compared with DMSO in MYH9 overexpression (∗P < 0.05 and ∗∗P < 0.01). (g) Correlation between MYH9 mRNA level and cell migration area in the U87MG cells.

We examined the RNA-seq data to see whether AHR was activated upon ITE binding. In expression levels of classical AHR target, cytochrome family members remained unchanged in ITE-treated group (Supplemental Table. 4). Gene set enrichment analysis was done to check the AHR pathway, which was not significant, either (Supplemental Figure. 4).

We further analyzed the relationship between MYH9 level and cell migration by treating MYH9 overexpressing cells with various ITE doses and measured their migration ability (Figure 4(g)). In addition to ITE's migration-inhibition effects in both groups, we found that MYH9 expression level correlated well with cell migration ability (Figure 4(h)), indicating that ITE's migration-inhibition effects were mediated by MYH9 at least partly.

4. Discussion

Drugs that can block cancer cell invasion are urgently sought after, and we discovered that ITE, an endogenous ligand of AHR, could block migration of the glioma cells ex vivo and in vivo, supporting ITE to be investigated as a lead compound. We identified a previously unknown target of AHR and MYH9, providing clues on how AHR regulates migration in human glioma cells. MYH9 is essential for both mesenchymal and amoeboid migration, and accordingly, we observed compromised cell migration and invasion. ITE's migration-inhibition effect had a nonmonotonic dose-response which was common in endocrine disruptors and has been seen in TCDD-treated cells [10]. Another factor that might contribute to the nonmonotonic dose-response was the tryptophan in the culture medium (25 μM), whose metabolite kynurenine is an AHR ligand. The affinity of kynurenine (32 μM) to AHR is lower compared to ITE (3 nM), and it is conceivable that ITE, especially at higher doses, dominated AHR's effects.

AHR either activates or inhibits cell migration, depending on the ligand and cellular context. AHR ligands, indirubin or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), activate the AHR pathway, upregulated the expression of AHR and CYPs, and promoted the renal cell carcinoma invasion by upregulating MMPs [11]. Activation of aryl hydrocarbon receptor promotes invasion of clear cell renal cell carcinoma and is associated with poor prognosis and cigarette smoke, Kynurenine, the endogenous ligand of the AHR, could either activate triple-negative breast cancer cell migration [12] or inhibit neuroblastoma cell migration to activate AHR [13].

Additional AHR ligands that have been reported for migration-inhibition effects include quercetin, omeprazole, indirubin, and 3-methylcholanthrene (3-MC). 20 μM quercetin inhibits U87 glioma cell invasion, 300 μM omeprazole inhibits breast cancer metastasis, 5 μM indirubin inhibits glioma invasion, and 1 mM 3-MC inhibits human umbilical vascular endothelial cell (HUVEC) migration. These ligands' effective concentration varies from several to hundreds of micromolar [14-17]. ITE inhibits glioma cell migration at nanomolar concentrations, exhibiting a significantly higher efficacy.

Various migration-related pathways were regulated by ligand-bound AHR, including phospholipase D1, CXCR4, and FAK. AHR' antagonist quercetin inhibited U87MG cells' invasion via downregulating phospholipase D1, omeprazole inhibited breast cancer metastasis in a mouse model and cancer patients via downregulation of the AHR-CXCR4 signaling pathway [14], indirubin inhibited glioma invasion in a mouse model via inhibition of GSK3beta [17], and AHR-agonist 3-MC inhibited HUVEC migration by reducing FAK activity.

We found that ITE inhibited migration and invasion via the AHR-MYH9 axis, supporting MYH9 as a novel AHR target. Neither classical AHR target cytochrome genes nor the AHR signaling pathway shown significant changes/enrichment. How AHR regulates MYH9 warrants further study.

Most of the observed pan class="CellLine">U87MG cell behavior upon ITE treatment could be explained by weakened actomyosin contraction due to lowered MYH9/NMIIA level. We observed compromised rear attraction, polarity, and mode switching in ITE-treated glioma cells in vitro. Cell spreading and protrusion dynamics are the net results of the centrifugal force generated by actin polymerization and the centripetal force generated by actomyosin contraction [18, 19]; so, with lower actin-myosin contraction resulted from lower MYH9, both cells spreading and filopodia extension increased, especially at the lateral side of the cell, as NMIIA inhibited lateral filopodia formation and maintained cell polarity. The weakened protrusion tip attachment probably resulted from lowered NMIIA too, as inhibition/knockdown NMIIA resulted in a reduction of the filopodia attachment strength [20], and inefficient lamellipodia attachment caused the ruffles [21]. Cell rear retraction depends on myosin IIA, which explained the inefficient rear retraction upon ITE treatment [22]. All these changes are similar to what was reported in cancer cells with depleted or lowered NMIIA [23, 24]. Moreover, the 20 hr ITE treatment caused cells to adopt a spherical shape, which was like what is shown in NMIIA knockdown cells after 12 hr in collagen matrix.

The inhibition of the mesenchymal to amoeboid transition in both 2D and 3D culture could have resulted from the lower pan class="Gene">MYH9 expression level upon ITE treatment. Cells need actomyosin contraction to become spherical [25]; hence, most of the ITE-treated cells remained in its original polygon shape. The inefficient blebbing amoeboid mode manifested as a lack of polarity is probably also due to the lower MYH9, which is needed to form a cell rear [26]. Blebs forming and retracting continuously in the round amoeboid migration, yet there is a lack of blebbing at the rear, which is essential for cell polarity.

Among the four possible cancer cell migpan class="Species">ration modes, we discovered that ITE inhibited both mesenchymal and the characteristic amoeboid A1 migration mode [27]. In GBM, to migrate in the brain, glioma cells have to squeeze through pores smaller than its cell body size, often with the help of matrix metalloproteinase where NMIIA is essential [28, 29] too. The reason for the lack of A1 mode in the ITE-treated group needs further investigation. Moreover, MYH9 promotes tumor progression by modulating the immune microenvironment, so reduced MYH9 level by ITE treatment potentially offers immune-promoting benefit in addition to its invasion-inhibiting effects [30].

When writing this paper, it is reported that programmed death-ligand1 (PDL1)/programmed cell death protein 1(PD1) blockade was effective as neoadjuvant therapy in GBM. The combination of ITE and PD1/PDL1 blockade warrants further research.

5. Conclusion

Small molecular AHR agonist ITE regulated glioma cells' migration via binding to AHR and inhibited the actin-myosin contraction due to reduced MYH9 protein level (Figure 5). ITE efficiently blocked glioma invasion in vitro, ex vivo, and in vivo in an orthotopic mouse glioma model.

Figure 5

ITE binds to its receptor AHR, which mobilized it to enter the nucleus and triggered MYH9 gene. Reduced MYH9 level caused lowered cellular contractility, which is required for both amoeboid and mesenchymal migration.