Clofazimine inhibits small-cell lung cancer progression by modulating the Kynurenine/Aryl Hydrocarbon receptor axis

Abstract

Small cell lung cancer (SCLC) is one of the highly metastatic malignancies that contributes to ~15% of all lung cancers. Most SCLC patients (50–60%) develop osteolytic bone metastases, significantly affecting their quality of life. Among several factors, environmental pollutant 2,3,7,8-Tetrachlorodibenzodioxin (TCDD) and kynurenine (Kyn), an endogenous ligand derived from tryptophan (Trp) metabolism, activate the aryl hydrocarbon receptor (AhR) and are responsible for SCLC progression and metastasis. Further, elevated AhR expression in bone cells intensifies bone resorption, making the Kyn/AhR axis a potential target for the bone metastatic propensity of SCLC. We first assessed the expression profile of AhR in human SCLC cell lines and found a significantly increased expression compared to normal lung cells. Additionally, we also evaluated the clinical significance of AhR expression in the patient samples of SCLC along with the relevance of the same in the Rb1^fl/fl; Trp53^fl/fl; Myc^LSL/LSL (RPM) mouse model using immunohistochemistry and found the higher AhR expression in the patient samples and RPM mouse tumor tissues. Using computational simulations, we found that clofazimine (CLF) binds at the activator (Kyn) binding site by forming a stable complex with AhR. The CLF binding with AhR was favored by Van der Waals and hydrophobic forces, and the proteins retained their secondary structure. Furthermore, we found that Kyn treatment potentiates the migration and clonogenic ability of SCLC cell lines by activating Erk/Akt oncogenic signaling. Blocking AhR with CLF reduces AhR expression, inhibits Kyn-mediated proliferation of SCLC cells, and induces apoptosis and cell cycle arrest in the G2/M phase; further, our examination indicates that Kyn treatment also promotes osteoblast-mediated osteoclast differentiation through RANKL. The treatment with CLF impedes RANKL expression and osteoclastogenesis, suggesting that CLF has the potential for developing SCLC therapies that have efficacies against bone metastasis.

Introduction

Lung cancer (LC) is a major contributor to cancer-related deaths worldwide and has the highest mortality rate globally [1]. In 30–40% of patients with advanced disease, metastasis to skeletal muscles remains the major challenge in lung cancer-related mortalities [2]. LC is mainly categorized into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), in which SCLC is highly aggressive in nature and is majorly associated with tobacco smoking [3]. Additionally, around 70% of SCLC patients develop distant metastases, including liver, bone, brain, and lungs, at diagnosis, and further bone metastasis, which is higher in SCLC patients compared to NSCLC [4]. According to a retrospective analysis of the Surveillance, Epidemiology, and End Results (SEERs) data, it was found that SCLC patients with bone metastasis have a drastically reduced survival rate from 13 to 9 months [5].

Several environmental pollutants and cigarette smoke are established as high-risk factors for lung adenocarcinoma, specifically, environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin), a highly toxic form of dioxide [6]. Several studies confirm that TCDD is a potent carcinogen that could activate different endocrine pathways via unique xenobiotic or aryl hydrocarbon receptors (AhR) [7]. Furthermore, it has been well established that AhR activity is crucial for various downstream effects that influence cell proliferation, formation of DNA adducts, tumorigenesis, EMT, and cancer progression, and correlates with poor patient outcomes [8]. A unique transcriptional signature of AhR in neuroblastoma has revealed its tumor-promoting involvement in MYCN-amplified neuroblastoma [9]. It has been shown that activated AhR can interact with retinoblastoma protein (Rb/E2F1 axis) and downregulate the S phage gene expression, resulting in a compromised cell cycle S phase in SCLC [10].

An important metabolite, Kyn, has been shown to aggravate the AhR activity and contribute to SCLC cell proliferation and disease progression [11]. Kyn is mainly produced from tryptophan catabolism, where indoleamine dioxygenase (IDO) converts the tryptophan into N-formyl-L-kynurenine, which further transforms into Kyn through formidase. Furthermore, IDO and tryptophan 2,3-dioxygenases (TDO) are recognized as rate-limiting enzymes for Kyn production and are often dysregulated in SCLC patients [12]. A recent study suggested that an increased expression profile of Kyn is associated with poor prognosis in SCLC patients [13]. SCLC is highly metastatic at an early stage, which contributes to it as a recalcitrant malignancy [14]. It has been shown that activated AhR can interact with retinoblastoma protein (Rb/E2F axis) and downregulate the S phage gene expression, resulting in compromised cell cycle S Phage. Moreover, bone tropism/skeletal metastasis can be correlated with the Kyn/AhR axis because bone cells such as osteoblasts (OBs) express AhR and interfere with SCLC-mediated bone modulation and niche formation by activating AhR in the bone microenvironment [15]. In addition, bone metastases is associated with poor prognosis in SCLC patients with limited therapeutic options [16]. Recently, riminophenazine-based, an FDA-approved anti-leprosy drug, Clofazimine (CLF), was identified as a potent antagonist and a suppressor of AhR. However, the antineoplastic activity of CLF in SCLC has not been explored. Therefore, the present research examines the functional aspect of the Kyn/AhR axis in SCLC-bone metastasis and inhibition of this axis with CLF to suppress the L-Kyn mediated growth and migration of SCLC cells [17]. AhR-dependent transcription induced by ligands is thwarted by CLF, which also inhibits AhR binding to DNA [18]. In two different studies (repurposing and in silico), CLF targets the Wnt/β-catenin signaling pathway, which significantly hampers the growth of triple-negative breast cancer cells and regulates the cancer stem cells plasticity in the complex treatment of glioblastoma [19, 20]. CLF acts as a potent inducer of apoptosis in chronic myeloid leukemia (CML) cells by intracellular ROS induction and suppressing the antioxidant enzyme peroxiredoxin 1. Further, an in vivo study revealed that CLF can target quiescent leukemia stem cells (LSCs) and does not harm normal hematopoietic stem cells [21]. In addition to SCLC, AhR and Wnt/β-catenin signaling promotes the OBs differentiation while preventing the differentiation of chondrocytes and adipocytes from MSCs [15, 22, 23]. Further, OBs secreted RANKL boost the differentiation of bone-reabsorbing cells osteoclasts (OCs) and initiate the SCLC-mediated vicious cycle in the bone microenvironment. Therefore, the impact of CLF on OBs’ and OCs’ differentiation has been investigated where CLF controls the bone-forming OBs and restrains the L-Kyn-mediated OCs activity.

Various functional studies were performed in this context to see the anticancer potential of CLF against SCLC cells, and we found that CLF has the potential to inhibit the proliferation, invasion, migration potential, and colonization ability of SCLC cells. The study revealed the anti-SCLC efficacy of CLF in high AhR-expressing cells. Also, it was seen that CLF binds to AhR in a competitive manner (molecular docking and dynamic simulations), thus inhibiting the L-Kyn-mediated colonization of SCLC cells through proliferative markers (Akt, Erk, FoxM1) and epithelial-mesenchymal transition (EMT) marker vimentin. CLF also regulates the L-Kyn-mediated OBs differentiation and consequently inhibits the bone resorptive functions of OCs.

Materials and Methods

Immunohistochemistry of human SCLC tissue microarray and RPM mice lung tissue:

BS04116a: human small cell lung carcinoma (SCLC) with lung tissue microarray, including TNM and clinical stage with 55 cases/100 cores, obtained from US bio max. This TMA contains 45 cases of small cell carcinoma from different stages, 10 lung tissues, duplicated cores of carcinoma, and a single core of lung tissue. Rb1^fl/fl; Trp53^fl/fl; Myc^LSL/LSL (RPM) mice lung tissues (n=5) were used to examine the status of AhR expression compared to the normal lung tissue using immunohistochemistry staining. In brief, the TMA and tissue slides were dried at 58°C overnight, and the next day, after keeping at RT for 15 min, four xylene washes were given. Then, the slides were rehydrated serially in 100–20% ethanol and rinsed with tap water. Further, slides were dipped into 3% H₂O₂ and rinsed with tap water. Next, after antigen retrieval by heating in sodium citrate buffer, slides were rinsed with tap water, subjected to primary AhR antibody overnight, and then incubated with HRP-conjugated secondary anti-mouse/rabbit IgG antibody for 30 min. After three PBST washes and 1 PBS wash, slides were subjected to DAB (3, 3 -diaminobenzidine) substrate kit (Vector Laboratories; SK-4100) and counterstained with hematoxylin. Then, the tissue slides were dehydrated serially in 20–100% ethanol and mounted after four xylene washes [24]. The graphs for AhR expression were plotted based on pathologist H-score evaluation and statistically evaluated using the unpaired t-test for RPM mice model and one-way ANOVA for TMA.

Molecular Docking:

To examine the affinity and binding site of CLF in AhR, molecular docking was conducted using AutoDock Vina [25]. The cryo-EM structure of the indirubin-bound Hsp90-XAP2-AHR complex with 2.85 Å resolution was retrieved from the PDB database (ID: 7ZUB). From this complex, the structure of AhR was extracted and used for molecular docking. Indirubin, an endogenous ligand and potent activator of AhR [26], was bound in this complex. First, the indirubin structure was separated from the complex and then redocked to validate the docking methodology. When redocked, indirubin occupied the same binding site and the same binding conformation as it was present in the reported cryo-EM structure (Figure 2A), verifying the docking method. To prepare the AhR structure, all non-protein atoms, such as water molecules, ions, indirubin, etc., were removed. The polar hydrogen atoms were placed to AhR, then Kollman charges were added [26]. The exhaustiveness used in molecular docking was 20, and spacing of grid was fixed at 1.00 Å to encompass the entire AhR molecule. The grid size was 60×44×64 Å with centre as x = 168.152, y = 168.935, and z = 166.725. The structures of CLF (CID: 2794) and Kyn (CID: 846) were taken from the PubChem database. The ligand structure was made flexible to obtain optimum binding conformations. After docking, only the conformation with the highest affinity was selected and used for analysis using PyMOL and Discovery Studio.

Figure 2. CLF interacts at the inhibitor/Kyn binding site in AhR.

A. Overlap of cryo-EM AhR-INDR complex with docked AhR-INDR complex. AhR is shown in rainbow ribbons, cryo-EM INDR is shown in red sticks, and docked INDR is shown in magenta sticks. B. Overlap of the cryo-EM INDR to docked CLF and Kyn in AhR. AhR is shown in rainbow ribbons, INDR is shown in red sticks, CLF is shown in green sticks, and Kyn is shown in magenta sticks. C. Two-dimensional representation of docked AhR-CLF complex. D. Two-dimensional representation of the docked AhR-Kyn complex. CLF is clofazimine, INDR is indirubin, and Kyn is kynurenine.

Molecular Dynamics Simulations:

The molecular dynamics (MD) simulation of apo AhR and its complexes with indirubin, CLF, and Kyn were conducted using the Gromacs 2018.1 packages to examine their stability, binding, and dynamics [27]. The MD simulations were done employing the Amber99sb-ILDN force field [28]. The topology of AhR was made in Gromacs, and the topology of ligands (indirubin, CLF, and Kyn) was prepared with Antechamber packages of AmberTools21 [29]. Both topologies were merged to make the complex’s topology. A triclinic box was created surrounding the structures where 1 nm distance was kept between the edge of the box and the structures to satisfy the periodic boundary conditions (PBC). The systems were then solvated by adding water molecules of the TIP3P model. First, the charges were neutralized by adding five chlorine atoms, and then 0.15 M NaCl was added to maintain the physiological salt levels. The energy of the systems was minimized using steepest descent algorithm before two sets of equilibration processes. The first set of equilibrations (NVT ensemble) was done with a V-rescale thermostat for 1000 ps (500000 steps) with 2 fs time step, where the volume, number of atoms, and temperature (310 K/37°C) were kept constant [30]. In the second equilibration phase, the system’s density and pressure were adjusted within the NPT ensemble. In this ensemble, the number of atoms, temperature, and pressure (1.0 bar) were held constant using a barostat (Parrinello-Rahman) for an additional 1000 ps (500000 steps) where time step was 2 fs [31]. The time constant in temperature coupling of both equilibrations was 0.1 ps. A 100 ns simulation was done at 310 K (37°C), and 5000 frames of each trajectory were saved. The trajectories were first corrected for PBC using -pbc cluster option in gmx trjconv. We only took protein/complex atoms as the output group for further analysis. All analyses were done using standard Gromacs 2018.1 modules. The components of binding energies between the ligands and AhR were computed using the g_mmpbsa package [32].

Cell lines and maintenance:

The human SCLC SBC3 and SBC5 cell lines were obtained from Osaka University and the Japanese Collection of Research Bioresources Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan, respectively. Human lung epithelial BEAS-2B and NCI-H69 SCLC cell lines were procured from ATCC (Rockville, MD, USA). Complete RPMI [HyClone RPMI 1640 with high glucose (4.5 g/L); Cat No.# SH30027.01] media containing 10% fetal bovine serum (FBS) supplemented with 1% penicillin-streptomycin (P/S) antibiotics (100μg/ml) and 1mM sodium pyruvate were used to maintain all cell lines at 37°C with 5% CO₂ in the humidifier atmosphere. All cell lines were maintained by replacing the fresh media on alternate days, and culture conditions were maintained as described earlier [33].

Primary culture of Osteoblasts and Osteoclasts:

Mouse calvarial osteoblasts (MCOs) were obtained from the primary culture of mouse calvaria using a standard protocol of sequential digestion with α-MEM containing 0.1% dispase and 0.1% collagenase [34, 35]. Briefly, pups of C57BL/6J mice (1 to 2 days old) from 3 different batches of animals were sacrificed by decapitation at 3 different time points (biological replicates), and calvarias were excised and pooled. After mincing and digestion, the released cells were collected in 10 ml FBS at the second to fifth digestions. Collected cells were centrifuged at 1200 rpm and seeded in T-25 flasks with complete α-MEM containing 10% FBS and 1% penicillin/streptomycin (complete growth medium). The MCOs were treated with different concentrations of L-Kyn and CLF in osteoblast differentiation medium comprising α-MEM supplemented with 10% FBS, 10 mmol/L β-glycerophosphate, and 5 mg/mL L-ascorbic acid). Conditioned media (CM) of MCOs were collected at 48 h, and culture conditions were maintained at 37°C with 5% CO₂ in the humidifier atmosphere, as described earlier [35].

Osteoclasts were cultured from 6 to 8-week-old C57BL/6 mouse bone (femurs and tibias) marrow-derived macrophages (BMMs) [24]. Briefly, bone marrows were flushed and cultured with 30 ng/mL macrophage colony-stimulating factor (MCSF) overnight in a CO₂ incubator. The next day, BMMs (1×10⁴ cells/well) were incubated with MCSF (30 ng/mL), and 50 ng/mL receptor activator of nuclear factor kappa beta ligand (RANKL) and treatment with OBs-CM (collected from control, L-Kyn, and L-Kyn + CLF) were done in 96 well tissue culture plate.

Cell viability and proliferation assay:

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction method was used to assess the viability and proliferation of SBC3 and SBC5 cells. Briefly, to see the impact of L-Kyn and CLF on SCLC cells, SBC3 and SBC5 cells were seeded in 96 well plates (3000 cells/well) and kept overnight in a complete RPMI medium containing 10% FBS supplemented with P/S and sodium pyruvate. The next day, SCLC cells were treated with various concentrations of L-Kyn (0–100 μM) and CLF (0–50 μM). After 48 h of treatment, MTT was added for two hours and incubated at 37°C in a CO₂ incubator. Finally, the MTT formazan crystals were dissolved in dimethyl sulfoxide (DMSO), and OD was measured at λ570 nm [36]. Cell proliferation and cytotoxicity were measured based on optical density.

Transwell migration assay:

To evaluate the migratory potential of L-Kyn and CLF on SCLC cells, 1×10⁶ SBC3 and SBC5 cells were plated in a 35 mm tissue culture dish and treated with L-Kyn (50 μM) and CLF (8 μM) alone and in a combination of both for 48 h. Untreated cells were taken as a control for each group. After treatment, both SBC3 and SBC5 cells were trypsinized and counted, and an equal number of cells were seeded onto Corning^® BioCoat^™ Matrigel^® Invasion Chamber, 8.0 μm PET Membrane in serum-deprived RPMI medium. However, 1 ml of 20% FBS containing RPMI was added in each lower chamber of 6 well plates. After incubation with L-Kyn and CLF for 48 h, migrated cells were fixed and stained with Diff-Quick stain. Migrated cells were photographed using an inverted microscope (EVOS FL Auto), the average number of cells per field was calculated, and the graph was plotted with the help of GraphPad Prizm software [37].

Colony formation assay:

To assess the colony formation ability of SCLC cells in the presence of L-Kyn and CLF, SBC3 and SBC5 cells (500 cells/well) were plated in 6 well plates with complete RPMI medium. The next day, both cells were treated with L-Kyn (50μM) and a combination of L-Kyn and CLF (50μM & 8μM). For 14 days, a fresh medium was added every two days. On the 15^th day, cells were washed with PBS, fixed with methanol, and stained with 0.5% crystal violet, as described previously [38]. Further, colonies were photographed with EVOS FL Auto, and the number of colonies was counted using the Image-J processing tool.

Flow cytometry for apoptosis and Cell cycle:

To quantify the apoptosis-inducing efficacy of CLF in SCLC cells, flow cytometry with Annexin V/PI staining was done. Briefly, SBC3 and SBC5 cells were trypsinized, and 1 × 10⁶ cells/well were seeded in 6 well tissue culture plates. Next day, the exhausted medium was replaced with a fresh medium containing L-Kyn (50μM) and a combination of L-Kyn and CLF (50μM & 8μM). After 48 h of incubation, control, L-Kyn, and L-Kyn + CLF treated cells were trypsinized, washed with PBS, and suspended in the 1X FACS binding buffer. Further, cells were incubated with propidium iodide (PI) and Annexin-V/CyTM5. After 15 minutes of staining, flow cytometric analysis was done using standard protocol. Cell cycle distribution analysis of SBC3 and SBC5 cells was done with propidium iodide (PI) containing Telford Reagent, and DNA content was measured using flow cytometry. In brief, both SBC3 and SBC5 cells (1 × 10⁶ cells/well) were seeded and treated in the same way as described in apoptosis analysis, and after 48 h of incubation, control, L-Kyn, and L-Kyn + CLF treated cells were trypsinized, washed twice with PBS and fixed with 70% ethanol overnight at 4 °C. Later, fixed cells were washed with PBS and incubated with Telford reagent for 1 h at 4 °C. Cell’s DNA contents were measured using flow cytometry, and cell cycle distribution was analyzed according to published methods [37, 39].

Western blot analysis:

The expression of specific regulatory proteins was determined through Western blot analysis. SBC3 and SBC5 cells were plated in 6 well tissue culture plates and treated with L-Kyn and L-Kyn + CLF for 24 h, as described in the above sections. After treatment, cell lysates were collected in RIPA buffer (10mM Tris-HCl; pH 8.0, 1mM EDTA, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1mM PMSF, and protease inhibitor cocktail). Cell lysates were collected after passing with a syringe and centrifugation at 4 °C for 30 min. A standard Bio-Rad DC Protein Assay kit was used to quantify the protein concentration in different cell lysates. After estimation, lysates were mixed with 6X protein loading dye (Tris-Cl; pH 6.8, SDS, bromophenol blue, glycerol, and β-mercaptoethanol), boiled at 95 °C for 10 min. Further, 20 to 40 μg/well protein from each sample was loaded and run on SDS-PAGE (8 to 16% according to the molecular size of the proteins), then transferred onto the PVDF membrane (100 V for 100 min). Next, blocking was done with a blocking solution (containing 5% non-fat milk and 0.1% Tween-20) for 1 h. Subsequently, membranes were incubated with primary antibodies (details are provided in table 1) overnight at 4 °C. Next day, membranes were washed thrice with PBST (1X PBS and 0.1% Tween-20), and then the blot was incubated for 1 h with their complementary secondary antibodies (1:5000 dilution) at RT. Again, the membranes were washed thrice with PBST, and the blots were developed using chemiluminescence ECL reagent (Pierce^™ ECL Western Blotting Substrate; Cat No.# 32106) exposed for 5 minutes [40, 41].

Table 1:

List of Antibodies used and details.

Antibodies	Dilution	Source	Identifier
Phospho-Akt	WB: 1:1000	Cell Signaling Technology	Cat#9271
Akt (pan)	WB: 1:1000	Cell Signaling Technology	Cat#9272
Bcl-xL	WB: 1:1000	Cell Signaling technology	Cat#2764
Cleaved PARP	WB: 1:1000	Cell Signaling technology	Cat#5625
FoxM1	WB: 1:1000	Cell Signaling technology	Cat#5436
Vimentin	WB: 1:1000	Cell Signaling technology	Cat#5741
AhR	WB: 1:1000	Invitrogen	Cat#MA1–514
Phospho-Erk1/2	WB: 1:1000	Cell Signaling technology	Cat#9101
Erk	WB: 1:1000	Cell Signaling technology	Cat#4695
RANKL	WB: 1:1000	Abcam	Cat#ab45039
β-Actin	WB: 1:1000	Cell Signaling technology	Cat#3700

RNA extraction and quantitative real-time PCR:

An RNeasy mini kit (Qiagen, #74106, Germantown, MD, USA) was used to extract the total RNA from OBs and OCs experiments. Extracted RNA samples were quantified by Nanodrop and 5X iScript RT supermix (Bio-rad, USA), and a total of 2 μg RNA was used for cDNA synthesis. Quantitative real-time PCR (qRT-PCR) was done using the SYBR^® Green Master Mix (Roche) and a Bio-Rad CFX96 Real-Time PCR Detection System. The relative mRNA expression of genes was calculated using average Δct value normalized to β-actin and represented as the fold changes compared with control. All primer details are provided in Supplementary Table S2 [42]. Statistical significance was calculated using 2-way ANOVA.

Alkaline phosphatase (ALP) and Alizarin Red S staining:

MCOs were treated with L-Kyn and L-Kyn + CLF and incubated for 2, 10, and 21 days. ALP, RT-qPCR, and Alizarin red staining (CPC-extracted Alizarin Red-S) were used to assess the effects of L-Kyn and L-Kyn+CLF on osteogenic differentiation of OBs. After treatment, OBs monolayer cells were rinsed with PBS, fixed in 10% formalin buffer for 1 min, and washed with Tris-buffered saline (TBS) without disturbing the monolayer of OBs. Then, enough BCIP/NBT substrate solution was added to cover the cellular monolayer and incubated at RT in the dark for 10–15 min. Subsequently, cells were washed carefully and evaluated for ALP staining results. Similarly, 21 days post-treatment, OBs were stained with Alizarin Red S staining solution and incubated at RT in the dark for 45 min. After staining, cells were washed four times with distilled water and visualized for OB mineralization (positive staining area indicating the calcified nodules). However, CPC-extraction of Alizarin Red-S staining was evaluated by using OD at λ 405. An unpaired t-test has been used to calculate the statistical significance of ALP and Alizarin Red S staining.

Tartrate-Resistant Acid Phosphatase (TRAP) Staining:

BMMs were treated with OBs-CM from control, L-Kyn, and L-Kyn + CLF and incubated for ten days. After treatment, OC cells were rinsed with PBS, fixed in 4% formaldehyde buffer for 10 min, washed with PBS, and then twice with distilled water. After washing, OCs were incubated for 1 h in TRAP staining solution at 37°C. Again, OCs were washed thrice with distilled water and photographed with an inverted microscope (EVOS FL Auto). The average number of TRAP^+ve cells per field was calculated, and the graph was plotted with the help of GraphPad Prizm software. An unpaired t-test has been used for TRAP counting.

Statistical Analysis:

GraphPad InStat software (GraphPad Software, Inc.) was utilized to calculate ANOVA or the student’s t-test for differences. P values below 0.05 were considered to be of statistical significance.

Results:

Overexpression of AhR has been associated with SCLC tumorigenesis in humans and mice:

Analysis of the AhR expression profile in three SCLC cell lines revealed that expression was substantially higher in SBC5, SBC3, and NCI-H69 cells compared to human normal bronchoalveolar epithelial cell line BEAS-2B, which is used as control (Figure 1A). To evaluate the clinical importance of AhR in SCLC patients, immunohistochemistry (IHC) was performed on tissue microarrays derived from patients, which included both SCLC samples and normal lung tissue. An increase in AhR expression has been observed in SCLC samples relative to healthy lung tissue, as shown in Figure 1B. Furthermore, a notably intense staining was detected within the cytoplasm of tissues obtained from patients diagnosed with SCLC. Additionally, a graphical depiction of IHC scoring indicates that elevated levels of AhR expression were correlated with more severe pathological grades (Figure 1B). To provide additional evidence in support of our hypothesis, we stained AhR in surgically resected SCLC samples obtained from RPM mice. These mice serve as a spontaneous model for SCLC and replicate conditions associated with human SCLC, including Rb1 and Trp53 inactivation and increased expression of the oncogenic form of MYC (MYCT58A) [43]. The IHC findings obtained from the animal model corroborate the results observed in the human samples. Notably, tissue samples originating from Rb1/Trp53/MycT58A exhibited pronounced AhR staining, in contrast to the lung tissue of control mice (Figure 1C). These findings demonstrated that AhR-mediated signaling may play a significant role in SCLC.

Figure 1. Enhanced expression profile of AhR is associated with an aggressive tumor phenotype in SCLC patients and in spontaneous mouse models.

A. The expression profile of AhR at the protein level has been shown in three SCLC cell lines (SBC3, SBC5, NCI-H69), and BEAS-2B, a normal bronchoalveolar epithelial cell line, was used as a normal control. β-actin served as a loading control. B. Representative images of IHC for AhR staining on tissue microarray with samples from SCLC patients and the normal lung tissue. Magnification x10 and x20 (left panel). Quantification analysis of IHC scoring of AhR in the tumor and normal lung tissue (right panel). C. Illustration showing strong staining of AhR in the lung of wild type and Rb1/Trp53/MycT58A mice (spontaneous mouse model for SCLC) (right panel) and quantification of IHC scores in the same (left panel). Results are represented as means ± SEM. Statistical significance has been calculated using one-way ANOVA and unpaired t-test. *p<0.05, ***p<0.0003, ****p<0.0001.

Clofazimine interacts at the activator binding site of AhR:

Before the molecular docking of CLF, the docking method was validated by taking out the activator of the AhR, indirubin, from the cryo-electron microscopy (cryo-EM) structure. This activator was found to be docked into the same position as it was present in the cryo-EM structure, thus confirming the validity of docking procedure (Figure 2A). Due to the absence of AhR-Kyn complex in PDB database, we docked AhR with Kyn and the docked structure served as control. Interestingly, CLF was docked at the active site of AhR (Figure 2B). The complex was primarily stabilized by the hydrophobic interactions, as shown in Figure 2C. Moreover, CLF also interacted with His337, Gly321, Thr289, Phe351, and Val363 of AhR through Vander Waals forces. Likewise, Kyn was also found to interact at the activator (indirubin) binding site (Figure 2B). The free energy for Kyn and AhR interaction was −7.6 kcal/mol, corresponding to the binding constant of 3.75×10⁵ M^-1. Hydrogen bonds involving Ser346, Ala367, Thr322, Cys333, Ser365, and Ser336 mainly stabilized the AhR-Kyn complex (Figure 2D). There was also involvement of hydrophobic forces (Ile325 and Phe295) and van der Waals forces (Gly321, Phe351, Val381, Ile349, Met340, and Ala334) in the complexation.

Clofazimine forms a stable complex with AhR:

To examine the stability and dynamics of CLF with AhR under physiological conditions, we performed MD simulation by mimicking (in the presence of 0.15 M NaCl and at 310 K/37°C) the physiologically relevant conditions. The preliminary analysis of the trajectories was done by examining the RMSDs (root-mean square deviations), as shown in Figure 3A. Apo AhR showed deviations till 40 ns, and then the trajectory became stable afterward. This indicates that the system became equilibrated roughly at 40 ns. A similar result was obtained for the AhR-indirubin complex. However, the deviations in the AhR-CLF complex were relatively lower. The average RMSD of apo AhR, AhR-CLF complex, AhR-indirubin complex, and AhR-Kyn complex were 1.232, 0.753, 1.141, and 0.809 nm, respectively. Further analysis was done by computing the RMSFs (root-mean square fluctuations) of C_α atoms of AhR without and with ligands (Figure 3B). The initial 15–20 residues of AhR exhibited larger fluctuations due to the coil form of this region, which tends to move freely in the aqueous environment. The RMSF of the majority of residues was less than 0.30 nm, which is considered relatively stable in the aqueous system. The average RMSF of apo AhR, AhR-CLF complex, AhR-indirubin complex, and AhR-Kyn complex were 0.279, 0.290, 0.267, and 0.229 nm, respectively, which show negligible changes in the average fluctuations. We also calculated the RMSF of all atoms of the ligands (supplementary Figure S1). There were some fluctuations in all ligands, which is attributed to the movement of ligands at the binding site.

Figure 3. CLF forms a stable complex with AhR.

A. Root-mean square deviations (RMSD) of backbone atoms of apo AhR, AhR-CLF complex, AhR-INDR complex, and AhR-Kyn complex as a function of time. B. Root-mean square fluctuations (RMSF) of C_α residues of AhR in the absence and presence of INDR, CLF, or Kyn. C. Radius of gyration (R_g) of backbone atoms of apo AhR, AhR-CLF complex, AhR-INDR complex, and AhR-Kyn complex as a function of time. D. Solvent accessible surface area (SASA) of apo AhR, AhR-CLF complex, AhR-INDR complex, and AhR-Kyn complex as a function of time. CLF is clofazimine, INDR is indirubin, and Kyn is kynurenine.

We also calculated the R_g (radius of gyration) and SASA (solvent-accessible surface area) as these parameters provide valuable insights regarding the changes in the compactness of proteins. The R_g of apo AhR decreased as the simulation progressed until 40 ns and became constant, as presented in Figure 3C. The decrease in R_g is an indicator of protein becoming more compact. A similar result was obtained for all systems. The average R_g of apo AhR, AhR-CLF complex, AhR-indirubin complex, and AhR-Kyn complex were 1.555, 1.588, 1.520, and 1.566 nm, respectively. The SASA of all systems with respect to simulation time is presented in Figure 3D. Like R_g, SASA also decreased until 40–50 ns and became constant. The decrease in SASA is due to the folding of AhR, which in turn decreased its solvent accessibility. The average SASA of apo AhR, AhR-CLF complex, AhR-indirubin complex, and AhR-Kyn complex were 98.757, 103.636, 95.325, and 98.094 nm², respectively.

Analysis of hydrogen bonding and effect of clofazimine’s binding on the secondary structure of AhR:

We analyzed the hydrogen bonding between the ligands and AhR. The number of hydrogen bonds made by the ligands with AhR throughout the trajectory is shown in Figure 4A. The continuous presence of hydrogen bonds was found in the trajectories of all complexes. The average of hydrogen bonds formed by CLF, indirubin, and Kyn with AhR was 0.802, 2.045, and 1.412, respectively. CLF formed lesser hydrogen bonds with AhR compared to indirubin and Kyn, further validating the molecular docking data where hydrophobic interactions mainly stabilized the AhR-CLF complex. The hydrogen bond existence map was also computed for all complexes. There was a lack of hydrogen bonds during the initial few nanoseconds in the AhR-CLF complex, and then hydrogen bonds became persistent (Figure 4B). Gly321 showed the highest hydrogen bond occupancy of 79.9% in the AhR-CLF complex. In the AhR-indirubin complex, two hydrogen bonds were persistent throughout the trajectory (Figure 4C). Two hydrogen bonds viz. Gln383 (96.4% occupancy) and Ser365 (97.7% occupancy) were found continuously in the trajectory of the AhR-indirubin complex. There were multiple pairs of hydrogen bonds in the trajectory of the AhR-Kyn complex Figure 4D. In the AhR-Kyn complex, Cys333, Phe295, and Arg318 showed the highest occupancies of 26.6%, 40.5%, and 44.3%, respectively.

Figure 4. CLF and Kyn form consistent hydrogen bonds with AhR throughout the trajectory.

A. The number of hydrogen bonds formed by CLF, INDR, or Kyn with AhR as a function of simulation time. B. Hydrogen bonds existence map of AhR-CLF complex as a function of time. C. Hydrogen bonds existence map of AhR-INDR complex as a function of time. D. Hydrogen bonds existence map of AhR-Kyn complex as a function of time. E. Average percentage of secondary structures in AhR in the absence and presence of CLF, INDR, or Kyn. CLF is clofazimine, INDR is indirubin, and Kyn is kynurenine.

We further examined the effect of the ligand’s binding on AhR’s secondary structure (Figure 4E). The average percentage of β-sheets, coils, bends, β-bridges, turns, α-helices, and 3’-helices in apo AhR were 26.10, 28.09, 9.78, 1.15, 15.35, 17.70, 1.80%, respectively. In the presence of CLF, there was a decrease in coils from 28.09% to 24.87%, while the percentage of α-helices increased from 17.70 to 19.82. This shows the partial stabilization of AhR’s structure following the complexation of CLF. Overall, there were fewer alterations in AhR’s secondary structure when complexed with either of three ligands (indirubin, CLF, or Kyn).

Analysis of principal components and energy minima lanscape:

We performed principal component analysis (PCA) to examine the flexibility of AhR without and in complex with ligands. Using PCA, we computed two principal components, i.e., eigenvectors. The projection of eigenvectors for apo AhR and the complexes are depicted in Figure 5A. The area occupied by all complexes (AhR-CLF complex, AhR-indirubin complex, and AhR-Kyn complex) was similar and comparable to apo AhR. Subsequently, eigenvectors were used to make free energy landscapes (FELs). Figure 5B–E illustrates the 3D FEL for AhR and its complexes. Remarkably, all systems reached the energy minimum, although their positions varied in the respective landscapes. Interestingly, only one energy minimum was found in apo AhR, AhR-indirubin complex, and AhR-Kyn complex. However, in the AhR-CLF complex, three distinct energy minima were obtained at 52.24, 55.66, and 64.58 ns of the trajectory. The energy minima complexes of Kyn and CLF were investigated by overlapping with the docked complexes (Supplementary Figure S2). It is interesting to note that both the complexes remained at the active site, further validating the stability of docked complexes. Further, the structures at energy minima were taken out and examined using Ramachandran plots, as shown in Supplementary Figure S3. In apo AhR and AhR-Kyn complex, only one amino acid was found in disallowed regions of the Ramachandran plot. However, in the case of the AhR-indirubin complex, no amino acid was present in the disallowed regions. All three energy minima structures of the AhR-CLF complex had no residues in the disallowed region.

Figure 5. Free energy landscapes of CLF and Kyn in complex with AhR.

A. Two-dimensional projection of eigenvectors of apo AhR, AhR-CLF complex, AhR-INDR complex, and AhR-Kyn complex. B. Three-dimensional plot of the free energy landscape of apo AhR. C. Three-dimensional plot of the free energy landscape of AhR-CLF complex. D. Three-dimensional plot of the free energy landscape of AhR-INDR complex. E. Three-dimensional plot of free energy landscape of AhR-Kyn complex. F. MM-PBSA binding energies for the interaction of CLF or INDR with AhR. CLF is clofazimine, INDR is indirubin, and Kyn is kynurenine.

To see whether the complexation of clofazimine is energetically favorable, the components of binding energy were calculated using MM-PBSA (Molecular Mechanics Poisson-Boltzmann Surface Area) method. Figure 5F illustrates the binding energies derived from MM-PBSA calculations. Notably, van der Waal energy (−55.55±0.29 kcal/mol) prominently drove the complexation of CLF with AhR, with a small contribution of the SASA energy (−5.98±0.02 kcal/mol). In the complexation of CLF to AhR, van der Waal forces were the main drivers. Polar solvation (41.41±0.38 kcal/mol) and electrostatic (11.72±0.38 kcal/mol) energies negatively affected the overall interaction of CLF. The binding of indirubin was mainly driven by van der Waals interactions (−38.03±0.26 kcal/mol) and partially by electrostatic energy (−9.83±0.15 kcal/mol) and SASA energy (−3.50±0.01 kcal/mol). Polar solvation energy (25.51±0.14 kcal/mol) also hampered the overall binding of indirubin. In AhR and indirubin complexation, van der Waals and electrostatics fixes were the majorly responsible for the interactions. The overall binding energies complexation of CLF and indirubin with AhR were −8.39 and −25.85 kcal/mol, respectively. The data from MM-PBSA calculations were further employed to obtain all complexes’ major energy-contributing residues in AhR (Supplementary Table S3). In the interaction between CLF and AhR, Asp329 (−4.62 kcal/mol) gave maximum energy contribution to overall binding followed by Glu312 (−4.48 kcal/mol), Glu335 (−4.22 kcal/mol), Glu415 (−4.08 kcal/mol), Asp425 (−4.02 kcal/mol), and Glu314 (−4.00 kcal/mol) Likewise, the highest energy contributing residues in complexation of indirubin with AhR were Phe295 (−1.61 kcal/mol), Val381 (−1.22 kcal/mol), Ile325 (−1.05 kcal/mol), Leu353 (−1.04 kcal/mol), Phe351 (−0.98 kcal/mol), Ile349 (−0.98 kcal/mol), His337 (−0.78 kcal/mol), and Ala367 (−0.50 kcal/mol). Overall, the outcomes of molecular docking and MD-simulation studies suggest that CLF binds to the active site of AhR, and Kyn binds at the activator site. The binding of CLF at the active site of AhR forms a stable complex, which could be a possible reason for inhibiting the activity of AhR.

Effect of CLF on Kyn-mediated SCLC proliferation, migration, and colonization of SCLC cells:

The proliferation of SCLC was subsequently investigated by subjecting the SBC3 and SBC5 cell lines to treatment with varying concentrations of Kyn for 48 h. The results indicate that a 50μM concentration of Kyn induces significantly enhanced cell proliferation in both cell lines, as depicted in Figure 6A. Furthermore, the cell proliferation induced by Kyn was considerably diminished when CLF, a well-established AhR inhibitor, was added to both cell lines. Notably, the IC₅₀ values did not exhibit a substantial change whether Kyn was present or absent (Figure 6B & C). To investigate the potential AhR-Kyn axis in SCLC cell migration and colonization to a greater extent, SBC3 and SBC5 cells were treated with Kyn in the absence and presence of CLF for 48h. It has been shown that treatment of Kyn substantially increased the colonization capabilities of both the SCLC cell lines along the number of migrated cells. At the same time, the presence of CLF causes a significant reduction in the Kyn-mediated colonization and migration potential of SBC3 and SBC5 cells (Figure 6D & E). To dissect the expression profile of Akt and Erk signaling (often activated by oncogenic mutations in many cancers) in SCLC cells, we performed the Western blot on SBC3 and SBC5 cells treated with Kyn alone or in the presence of CLF. It was observed that proteins (such as Akt and Erk) involved in cancer cell growth and survival are highly expressed and phosphorylated in the Kyn-treated SCLC cells along with the higher expression of vimentin, a known marker for endothelial-to-mesenchymal transition. Interestingly, inhibition of AhR in SBC3 and SBC5 with the treatment of CLF, even in the presence of Kyn, significantly inhibits the phosphorylation of Akt and Erk along with the downregulation of FOXM1 and vimentin, as shown in Figure 6F. These results suggest that AhR may present a viable target in SCLC. AhR inhibition leads to a substantial decrease in cancer cell proliferation, colonization, and migration, along with the downregulation of oncogenic signaling.

Figure 6. CLF treatment blocked the Kyn-mediated cellular proliferation, migration, and colonization in SBC3 and SBC5 cell lines.

A. MTT assay was used to measure the effect of Kyn on cell proliferation. 50μM of Kyn treatments showed a significant increase in the proliferation of SCLC cell lines. B&C. Cytotoxicity profile of CLF has been shown through MTT assay on SBC3 and SBC5 cell lines in the presence and absence of 50μM Kyn. CLF substantially inhibits the Kyn-mediated proliferation in both the SCLC cell lines without showing any significant increase in the IC₅₀ values. The IC₅₀ value of the drug was calculated using GraphPad Prism^®7 software. D&E. Migration and colonization inhibiting capability of CLF in the presence of 50 μM Kyn has been studied by performing trans well migration and colony assays on SBC3 and SBC5 cell lines. Representative images (left panel) illustrating enhanced migration and colonization of SBC3 and SBC5 cells in the response to Kyn treatment. However, CLF treatment strongly inhibits both. Crystal violet was used to stain migrated cells with the imaging of five different areas at a magnification of 10x. Scale bar = 100μm. Representative bar graph for the quantification of cell numbers showing migration and colonization in control, 50 μM Kyn treated and 8 μM CLF with 50 μM Kyn treated samples (left panel). Data are shown as means ± SEM. F. The expression levels of oncogenic proteins involved in Akt/Erk signaling have been elucidated with western blotting of SBC3 and SBC5 cells after 48h treatment of 50 μM Kyn in the presence or absence of 8 μM CLF. β-Actin was used as a loading control. ****p<0.0001, ####p<0.0001 (compared to Kyn’s treatment).

Effect of CLF on apoptosis and cell cycle progression in SCLC cell lines:

Cellular proliferation and apoptosis equilibrium disruption are widely recognized as the principal factors in determining cancer etiology. Therefore, we have investigated the possibilities of AhR on the apoptosis of SCLC cell lines SBC3 and SBC5. Interestingly, in both cell lines (SBC3 and SBC5), FACS studies revealed no significant increase in the number of live cells in Kyn-treated samples compared to the control group. However, the treatment of CLF substantially increased the percentage of apoptotic cells to approximately 65% in both the cell lines, even in the presence of Kyn (Figure 7A). The gating strategy has been shown in Supplementary Figure S4.

Figure 7. Effects of CLF in the presence of Kyn on inducing apoptosis and G2/M cell cycle arrest in small cell lung cancer cells.

A. The number of apoptotic cells was analyzed through flow cytometry analysis in SBC3 and SBC5 cell lines after their treatment with Kyn alone or in the presence of CLF for 48h (left panel). Quantification of apoptotic cells in control, 50μM Kyn treated and co-treatment of 8μM of CLF with Kyn for 48h. Student t-test has been used to identify statistical significance ****p<0.0001, and ####p<0.0001 (compared to Kyn’s treatment).B. SBC3 and SBC5 cell lines were subjected to DNA content analysis through FACS in various cell cycle phases by propidium iodide staining. The representative FACS histogram from three independent experiments shows the potential of CLF in the presence of Kyn to arrest the SCLC cells in the G2/M phase (left panel). A representative bar graph of quantitative analysis of DNA content was shown as mean ±SD (n=3) (right panel). The presence of CLF significantly arrests the cell cycle progression in the G2/M phase. C. Western blot analysis of Bcl-xl and cleaved caspase 7 shows that treatment of CLF in the presence of Kyn increased the expression of apoptotic marker protein with a significant reduction in the expression of antiapoptotic protein. Β-Actin was used as a loading control. One way ANOVA was employed for statistical significance **p<0.001, ****p<0.0001, ##p<0.001, ####p<0.0001 (compared to Kyn’s treatment).

Next, we examined the cell cycle distribution through FACS to dissect the probable mechanism involved in the CLF-mediated inhibition of SCLC proliferation. Our data revealed that 8μM of CLF could suppress cell growth as compared to control and Kyn-treated samples and resulted in an exceptional cellular accumulation in the G2/M phase, which was accompanied by a significantly decreased DNA content in S and G0/G1 phase in SBC and SBC5 cells (Figure 7B). These observations suggest that the AhR might function as a regulator of apoptosis and cell cycle in SCLC and enhance lung cancer tumorigenicity. We performed the Western blot to further analyze the protein expression patterns associated with the apoptotic pathway. We found that CLF-treated samples have lower and higher anti and proapoptotic protein expression, Bcl-xL, and cleaved PARP, respectively, as shown in Figure 7C.

The Kyn/AhR axis is crucial in small-cell lung cancer-mediated differentiation of osteoblasts:

As AhR is well known to be present in OBs [44], to examine whether the Kyn/AhR axis is functionally important in mediating the SCLC-mediated bone cell differentiation, we performed functional studies on MCO with Kyn in the presence or absence of CLF as shown in figure 8A. Figure 8B & C shows that treatment of 50μM Kyn significantly enhanced the ALP activity and staining. At the same time, the co-treatment of CLF with Kyn showed a remarkable decrease in both parameters. Furthermore, we have also performed the expression profile of osteogenic genes through RT-PCR, and our data indicates that samples treated with Kyn have higher mRNA expression of both early and late osteoblastic markers ALP, OC, and Col-1a-1, respectively, compared to control. However, treatment with 8μM of CLF significantly inhibits Kyn-mediated osteoblastic differentiation in SCLC (Figure 8D). Additionally, the influence of Kyn on the mineralization of OBs was examined due to the fact that differentiated MCOs have the ability to mineralize bone matrix. As evident in Figure 8E & F, MCOs treated Kyn showed strong alizarin red-S-stained nodules as compared to the control, while the treatment with CLF significantly abrogated the staining. In aggregate, all these results conclude that the Kyn/AhR axis has been involved in mediating bone resorption in the case of SCLC. Since OBs regulate OCs’ function through RANKL, we performed the Western blot on MCOs treated with Kyn alone and in the presence of CLF. We found that the sample treated with Kyn showed a notably increased manifestation of RANKL compared to the control. However, CLF treatment substantially inhibits Kyn-mediated RANKL expression, as shown in Figure 8G.

Figure 8. CLF inhibits the Kyn-mediated differentiation of osteoblasts.

A. Schematic representation of OBs culture, treatment, and collection of conditioned media. B & C. Kyn treatment in undifferentiated MCOs promotes osteoblast differentiation, which was inhibited by the treatment of 8 μM CLF. Undifferentiated MCOs were treated with 50 μM of Kyn in the presence and absence of 8μM CLF for 2 and 10 days and were checked for alkaline phosphatase activity and staining. D. Undifferentiated MCOs were treated with 50 μM of Kyn in the presence and absence of 8μM CLF for 48h in the differentiated medium of osteoblast, followed by the extraction of total RNA and RT-PCR analysis for osteogenic genes (ALP, Runx-2, Col-1a, and osteocalcin), and gene expression was normalized to that of β-actin. E. Representative images of Alizarin staining showing strong mineralization in the MCOs treated with 50 μM of Kyn. However, the presence of 8 μM CLF significantly inhibits the same. F. Quantification of alizarin staining in the form of OD values. G. Western blot analysis of RANKL expression in the MCOs treated with the 50 μM of Kyn in the presence and absence of 8 μM of CLF in the differentiated osteoblast medium for 48h. β-Actin was used as a loading control. Results are expressed as mean ±SD. Unpaired t-test and two-way ANOVA were used to measure statistical significance. ****p<0.0001, ####p<0.0001 (compared to Kyn’s treatment).

The Kyn/AhR axis mediates osteoclast differentiation:

We further study whether the secretome of OBs treated with Kyn alone or in the presence of CLF has an osteoclastic nature, as shown in Figure 9A. Therefore, we exposed the BMMs to the 40% conditioned medium of OBs treated with Kyn in the presence or absence of CLF. Our RT-PCR data revealed that BMMs culture treated with the conditioned medium derived from only Kyn-treated OBs has a significantly higher expression of osteolytic markers like TRAP, cathepsin K, NFATc1, c-Anhydrous, and c-fos (4, 4, 3, 7, and 6 folds respectively) in comparison to the cells treated with the secretome of controlled OBs. In contrast, the BMMs treated with the conditioned medium of OBs co-treated with Kyn and CLF showed substantial downregulation of these genes at RNA levels (Figure 9B). Additionally, TRAP staining also compliment the RT-PCR data by revealing a similar observation for OC differentiation, which demonstrates that BMMs cells exposed to conditioned medium derived from only Kyn-treated OBs showed significantly higher TRAP^+ve cells as compared to control while the BMMs exposed to CLF co-treated medium has significantly lower number of TRAP^+VE cells (Figure 9C).

Figure 9. CLF treatment inhibits the osteoblast-mediated osteoclast differentiation.

A. Schematic representation of OCs culture and treatment with OBs-CM. B. BMMs derived from C57BL/6J mice were subjected to culture in the presence of 40% CM derived from osteoblasts treated with 50 μM of Kyn alone or in the presence of 8 μM of CLF for 48h with MCSF. RNA was extracted, followed by an RT-PCR analysis of osteolytic genes like TRAP, Cathepsin K, NFATc1, c-Anhydrase, and c-fos. β-actin was used to normalize the gene expression. C. Representative images of TRAP after 7 days showing strong staining on BMMs treated with 40% condition medium of only 50 μM of Kyn treated cells as compared to control, while the treatment of condition medium derived from osteoblasts received of 8 μM of CLF along with 50 μM of Kyn significantly reduced the staining (left panel). Quantification of TRAP^+VE cells in the form of a bar graph. Data are shown as means ± SD. Two-way ANOVA and unpaired t-tests have been used for statistical significance. ***p<0.0004, ****p<0.0001, ####p<0.00001(compared to Kyn’s treatment).

Discussion:

SCLC is a highly metastatic and recalcitrant cancer which severely hampers the survival of the patients. Specifically, bone metastasis significantly reduces the overall survival rate of SCLC patients. The precise mechanism behind aggressive and multisite metastases is not known so far. Still, numerous studies have emphasized some altered proteins that could participate in the bone metastatic process [45, 46]. Recently, the treatment paradigm for lung cancer has shifted towards targeted therapies, which specifically inhibit the expression pattern of molecular targets that are critical for the proliferation and survival of cancer cells. These targets might be cell surface receptors or any intracellular molecule of the signaling cascade. Numerous known targeted therapies exist, like the use of erlotinib and gefitinib, which act as an inhibitor of epidermal growth factor receptor (EGFR). Furthermore, crizotinib and alectinib are known to inhibit the expression of anaplastic lymphoma kinase (ALK). The success of targeted therapies over conventional first-line chemotherapy is evident from the fact that the induction of EGFR inhibitors has a 42.6% increase in efficacy, which ultimately led to a significant improvement in the overall survival rate of patients [47]. However, the utility of these therapies in clinics in strictly rely on the presence of the target molecule within the tumor. If the particular target is not critical for the oncogenesis process, then the drug’s therapeutic potential might be absent or limited [48]. This study elucidates the involvement of the Kyn/AhR axis in the SCLC’s advancement and metastasis, and the addition of Kyn modulates the function of bone cells. Treatment of L-Kyn significantly enhances the proliferation and migration of SCLC cells through the activation of AhR-mediated oncogenic signaling. Similarly, following L-Kyn treatment, bone cells showed a boost in differentiation and functions in vitro. Therefore, the Kyn/AhR axis is crucial for both SCLC and bone cells, and targeting this axis with CLF could be critical to control the proliferation and metastasis, which ultimately prevents the bone metastatic and niche formation capability of SCLC cells in the bone microenvironment. Recent reports indicate the high significance of AhR signaling in the SCLC development induced by human papillomavirus-16 E6/E7 oncoproteins and tobacco smoke interactions [10, 49]. Likewise, altered tryptophan metabolism, Kyn production, and AhR activation are associated with immune modulation and metastasis in various malignancies like melanoma, hepatocellular carcinoma, and colorectal cancer [50–53]. A study conducted by Li et al. established that the PLK1-mediated phosphorylation of AhR leads to metastasis in patients of lung adenocarcinoma through the activation of the DIO₂-TH signaling cascade [54]. Significantly increased AhR expression in SCLC cells relative to normal cells indicates that the Kyn/AhR axis is involved in the development and progression of this disease, as demonstrated by our findings. Moreover, elevated AhR expression facilitates the advancement of SCLC.

Considering the role of Kyn in cancer, especially in the proliferation and metastasis of SCLC to bone, we have tried to explore the possibilities of inhibiting the Kyn/AhR axis. The AhR functions as a ligand-activated transcription factor, which can suppress immune signaling and ultimately contribute to immune dysfunction and the advancement of cancer [55]. Indirubin, a naturally occurring ligand of AhR derived from the diet and synthesized from tryptophan by intestinal bacteria [56], has been identified through cell-based assays as a potent activator of AhR [57]. Due to the unavailability of the complex structure of AhR with Kyn, we have used the AhR-indirubin complex. Moreover, we have also taken the docked complex of Kyn with AhR as control. Molecular docking studies have demonstrated that Kyn and CLF bind at the active site or ligand-binding pocket in AhR, where indirubin binds. Indirubin interacts with Phe352, Ile349, Cys333, Ser365, Leu353, Gln383, Ala367, Phe295, His337, Val381, His291, Pro297 of AhR’s ligand-binding pocket [57]. CLF also interacts with the majority of the residues of ligand-binding pockets. The binding of CLF is expected to prevent the complexation of indirubin or Kyn, which is required for downstream signaling.

We further examined the energetics of the interaction. Typically, in protein-ligand interactions, the non-covalent forces like electrostatic interactions, hydrophobic interactions, van der Waals forces, and hydrogen bonds predominate, collectively influencing the overall binding. In the complexation of CLF with AhR, van der Waal energy prominently drove the complexation, with a minor influence from SASA energy. Through molecular simulations of the cryo-EM structure, it has been observed that indirubin maintains stability in its experimental position. Furthermore, its presence stabilizes the bound conformation of AhR, establishing stable contacts with most residues noted in the cryo-EM structure. Notably, most amino acids within the ligand-binding pocket form favorable interactions with indirubin [57]. All these data provide sufficient evidence to target the AhR using CLF. Further, the efficacy of CLF was validated in cancer cells.

Numerous reports have considered L-Kyn, which is produced from altered metabolism and acts through AhR, augmenting the proliferation, stemness, metastasis, and immune evasion of cancer cells [11, 58, 59]. Therefore, this study reveals the functional consequences of L-Kyn with respect to the proliferation, migration, and colonization potential of SCLC cells. We found that L-Kyn supplementation supports the growth and aggression of SCLC cells. Various research studies revealed that dysregulation in Akt signaling cascades due to the amplifications or mutations in the genes related to this pathway, like loss of PTEN or activation of receptor tyrosine kinases (RTKs). This disturbance plays a key role in driving tumor progression and metastasis by promoting survival and inhibiting the process of apoptosis [60]. Moreover, recent mechanistic studies have revealed that activation of AhR triggers one or more components of FOXM1/Akt/Erk downstream signaling for the proliferation, tumor progression, and metastases of several cancers [61–66]. For instance, a study conducted by Wang et al. on lung cancer identifies that interleukin-8 mediated proliferation and tumor progression involves Akt/Erk signaling axis through the activation of the epidermal growth factor receptor [67]. Similar events have been observed in this study, where L-Kyn treatment elevates the FOXM1 expression through Ahr binding and subsequently increases the phosphorylation of Akt and Erk in SCLC cell lines. However, CLF, in the presence of L-Kyn, potentially reduced the expression and phosphorylation of the same axis signaling molecules. Our results are in accordance with the previous reports that inhibition of FOXM1-mediated signaling improves the therapeutic outcome of immunotherapy in lung cancer cells [68].

Furthermore, EMT is a critical phenomenon that fuels cancer cells’ invasive and migratory potential [69]. Recently, it has been reported that AhR promotes the EMT of esophageal squamous cell carcinoma, and modulation of AhR through 3,3′-Diindolylmethane reverses the EMT [70]. We found that mesenchymal marker vimentin is increased with L-Kyn and extensively decreased with CLF treatment in SBC3 and SBC5 cells. Therefore, the AhR functions are well established in SCLC cells’ proliferation, migration, colonization, and EMT by altering the signaling molecules FOXM1, Akt, Erk, and vimentin. Despite several investigations of CLF action against cancer cells in vitro, few studies highlighted the novel mode of action to induce apoptosis and cell cycle arrest [21, 71, 72]. For instance, a study conducted on pancreatic ductal adenocarcinoma (PDAC) using a SCID mouse mode found that CLF substantially increases survival rates by reducing tumor growth through the induction of apoptosis [72]. Likewise, in our assessment, CLF showed significant capability to impede SCLC growth in two distinct methods: by inducing significant apoptosis and cellular arrest during the G2/M phase of the cell cycle. Moreover, CLF treatment downregulates the antiapoptotic gene Bcl-xL and increases the cleaved-caspase7 in SBC3 and SBC5 cells, which supports the apoptosis-inducing capability of CLF in SCLC.

In advanced stages, osteolytic bone metastasis is more prominent in the case of SCLC, where the vigorous activity of OCs governs bone remodeling [35, 73–75]. Even so, the differentiation of OCs from hematopoietic stem cells (HSCs) is majorly controlled by OBs through RANKL and OPG equilibrium [76–78]. Furthermore, AhR-mediated signaling has been revealed in OBs and implicated in the regulation of skeletal progenitor cells [15]. These results suggest that it’s possible that SCLC-secreted L-Kyn can boost the OBs differentiation, followed by secretion of osteoblastic RANKL, and consequently create osteolytic bone lesions by exacerbating the OC’s functions [79–81]. However, there are contradictory reports also which show that Kyn has an inhibitory effect on the formation of osteoblasts and eventually leads to bone cells. For instance, in vitro and in vivo studies conducted by Lawrence et al. found that higher levels of Kyn cause suppression of mitochondrial metabolism in bone cells and subsequently result in a negative impact on osteoblast function by reducing their energy production [82]. Some clinical observational studies also found that IFN-γ-mediated inflammation and high Kyn levels downgrade bone mineral density (BMD) and consequently increase the risk of fracture in the elderly population [83, 84]. We observed that the RANKL expression is increased after the addition of L-Kyn to the OBs, and this L-Kyn/AhR axis enhances the OBs differentiation and function. However, when OC progenitor cells come into the vicinity of these active OBs, the differentiation and function of OCs increase; consequently, by increasing the OC’s functions, SCLC cells are easily able to make their niche in the microenvironment of bone and create the osteolytic bone lesions. However, the CLF treatment controls both the proliferation of SCLC cells as well as the L-Kyn-mediated differentiation of OBs by blocking the L-Kyn/AhR axis in the bone microenvironment. Therefore, CLF could prevent SCLC progression and bone metastatic events in a systemic setting.