Erianin serves as an NFATc1 inhibitor to prevent breast cancer-induced osteoclastogenesis and bone destruction

Graphical abstract

Highlights

• •Erianin significantly alleviates breast cancer induced osteolysis in vivo.
• •62.5–250 nM erianin inhibits 231 CM and RANKL activated osteoclastogenesis.
• •Erianin interacts with NFATc1 but not SRC protein to suppress osteoclast generation.
• •Erianin suppresses excessive osteoclast formation in breast cancer patients.
• •Erianin promises as a novel treatment as an NFATc1 inhibitor of breast cancer-induced SREs.

Abstract

Introduction

Breast cancer-related bone metastasis can lead to skeletal-related events (SREs), which decrease patient quality of life. Inhibition of osteoclastogenesis is a key treatment for SREs; however, the availability of clinical drugs remains limited, and all existing ones disrupt physiological bone formation, while exhibiting no effect on patient survival time.

Objectives

This study aimed to identify a novel osteoclast inhibitor for the treatment of breast cancer-induced SREs.

Methods

The MDA-MB-231 breast cancer cell-induced bone loss model was used to investigate the therapeutic effects of erianin in vivo. Then, we evaluated the inhibitory effects of erianin on osteoclastogenesis and signalling in bone marrow-derived macrophages (BMMs) induced by conditioned medium from MDA-MB-231 breast cancer cells (231 CM) and receptor activator of nuclear factor-κB ligand (RANKL) in vitro. Next, a Cellular Thermal Shift Assay and siRNA-mediate knockdown were performed, to investigate the target of erianin during osteoclast formation. The effects of erianin on human osteoclastogenesis were evaluated using CD14⁺ monocytes obtained from patients with breast cancer.

Results

Erianin effectively improved breast cancer cells-induced bone destruction at doses of 2 and 20 mg/kg/day in vivo, while suppressing osteoclastogenesis and the upregulation of SRC-NFATc1, INTEGRIN β3-MMP9 signals induced by 231 CM and RANKL in vitro. Furthermore, erianin interacted with NFATc1 but not SRC, and Nfatc1 knockdown eliminated the inhibitory effects of erianin on osteoclastogenesis. Notably, lower expression of NFATc1 positively correlated with longer survival in patients with cancer and a high risk of bone metastasis. We further revealed that 62.5–250 nM erianin suppresses NFATc1 and excessive osteoclastogenesis in CD14⁺ monocytes from patients with breast cancer.

Conclusion

Erianin acts as an NFATc1 inhibitor that attenuates breast cancer-induced osteoclastogenesis and bone destruction.

Introduction

Bone metastasis occurs in approximately two-thirds of patients with stage II/III breast cancer, leading to various skeletal-related events (SREs), including hypercalcemia, pathological fractures, and severe bone pain [1]. The occurrence of SREs significantly affects the health status and expected lifespan of patients with metastatic breast cancer, and the 5-year survival rate of patients with bone metastasis decreases from 8.3% to 2.5% when combined with SREs [2]. The delicate equilibrium between osteoblast- and osteoclast-mediated bone formation and resorption, plays a vital role in maintaining healthy bone density and essential physiological functions [3], [4]. In recent years, osteoclast inhibition has been recognized as an effective strategy for mitigating bone destruction and other SREs caused by bone metastatic cancer cells [5], [6].

Aberrant osteoclast differentiation in breast cancer is triggered through both RANKL-dependent and-independent pathways. During the initial stages of classical osteoclastogenesis, RANKL binds to its ligand RANK, triggering the activation of nuclear transcription factor NF-κB and the c-FOS signal, which subsequently induces the early expression and nuclear translocation of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) [7], [8], [9]. Furthermore, the phosphorylation of the SRC kinase enhances Ca²⁺ oscillations and ultimately promotes the nuclear translocation of NFATc1 [10], [11]. During the middle and late stages of osteoclastogenesis, NFATc1 stimulates the expression of genes crucial for osteoclast development, such as Nfatc1, c-Src, Integrin β3, matrix metalloproteinase 9 (Mmp9), tartrate resistant acid phosphatase (Trap), and cathepsin K (Ctsk); culminating in the formation of mature osteoclasts [12]. The activation of the INTEGRIN β3-MMP9 signal promotes the formation of the F-actin ring, and helps maintain the bone resorption function of mature osteoclasts [13], [14], [15]. Breast cancer bone metastasis disrupts the equilibrium between bone formation and resorption by generating various cytokines, including IL-6, TNF-α, and IL-11, etc. These cytokines induce RANKL-independent osteoclastogenesis, which is particularly significant in the pathological bone loss induced by breast cancer bone metastasis [5], [16].

Current drugs used to treat breast cancer-induced SREs via targeting osteoclasts include bisphosphonates and the monoclonal anti-RANKL antibody, denosumab [17], [18], [19]. Nevertheless, the extended use of bisphosphonates and denosumab potentially leads to jawbone necrosis and rebound effects, which may be related to the suppression of normal osteoclast production and signalling pathways [20]. Furthermore, triple-negative breast cancer exhibits the secretion of many inflammatory factors, such as IL-11, which induces RANKL-independent pathological osteoclastogenesis, thereby limiting the effectiveness of denosumab [5]. Other studies have shown that bisphosphonates and denosumab do not prolong the overall survival of patients with breast cancer with bone metastases [21]. Therefore, there is an urgent need for novel therapeutic agents that can inhibit breast cancer-induced osteoclastogenesis while offering improved risk–benefit profiles.

Erianin, derived from Dendrobium chrysotoxum, is a natural product that exhibits various beneficial properties, such as anti-tumour, anti-inflammatory, and anti-infective effects [22], [23], [24], [25], [26]. It has been reported to inhibit the progression of several tumours, e.g., osteosarcoma [22], breast cancer [23], melanoma, colorectal cancer [24] and lung cancer [25], at low doses both in vitro and in vivo. In addition, erianin has shown therapeutic potential in CIA mouse models, as well as against inflammation and gout [26]. However, the precise role of erianin in osteoclastogenesis and its effect on breast cancer-induced SREs remain unclear.

In this study, we report that erianin is an NFATc1 inhibitor that effectively suppresses the formation of osteoclasts and osteolytic damage induced by breast cancer. This suggests that erianin serves as a potential inhibitor of osteoclasts during healing of breast cancer-induced bone destruction and other SREs.

Materials & methods

Antibodies and reagents

Erianin (Fig. 1A) was obtained from Chengdumust (Sichuan, China). Australian FBS, α-MEM and DMEM were provided by Gibco (NY, USA). The TRAP staining kit (387A-1kT), CCK-8 kit, LPS (055: B5), and DAPI were obtained from Sigma (MO, USA). The M-CSF and RANKL were provided by PeproTech EC, Ltd (NJ, USA) and R&D Systems (MN, USA), respectively. The MTT, RNA isolation kit, Luciferase reagents, PrimeScript RT reagent kit and real-time fluorescence quantitative reagent kit, were purchased from Promega (WI, USA). The hydroxyapatite-coated plates were obtained from Corning (NY, USA). Rhodamine-conjugated phalloidin (40734ES75) was purchased from Yeasen Biotech (Shanghai, China). Antibodies for NFATc1 (D15F1), MMP9, INTEGRIN β3, SRC and p-SRC, were provided by Cell Signaling Technology (MA, USA). The β-Actin antibody and DAPI were obtained from Beyotime Biotechnology (Guangzhou, China).

Fig. 1.

Erianin ameliorates breast cancer cell-induced bone loss in vivo (A) The chemical structure of erianin. (B) Specific modeling process. (C) Body weight record (n = 6). (D) Representative 3D micro-CT reconstructed images of trabecular bone. (E–F) The microstructural parameters including BMD, Tb.N, BV/TV, Tb.Th, Tb.Sp, SMI, BS/BV and Conn.D (n = 6). (G) H&E histology and TRAP staining of the tibia. (H) Quantitative analysis results of bone sections, including N.Oc/BS, N.Oc, Oc.S/BS (n = 6). The results are presented as mean ± SD (^## p < 0.01, ^#### p < 0.0001 vs Sham group; * p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001 vs Model group).

Animal models and analytical methods in vivo

Female 6–7-week-old BALB/c nu/nu mice, were provided by the Guangdong Medical Laboratory Animal Center. The housing conditions of the mice and the breast cancer MDA-MB-231 cell-induced bone destruction mouse model were established as previously reported [27]. Eight-week-old mice were randomly assigned to four groups (n = 6 each): sham plus 1% DMSO, model plus 1% DMSO, model plus 2 mg/kg erianin, and model plus 20 mg/kg erianin. Then 50 μL of MDA-MB-231 cells (5 × 10⁶/ mL) was injected into the mouse tibial in the model group, with PBS being used for the sham group, following surgery. Intraperitoneal injections of erianin at doses of 2 and 20 mg/kg were administered daily for 18 days, and the mice were harvested after the operation, as illustrated in Fig. 1B. The tibias were fixed in 4% PFA and analysed using a SkyScan 1072 Micro CT System. Subsequently, the tibias were decalcified using 12% EDTA for one month. The decalcified tibias were embedded in paraffin for haematoxylin and eosin (HE) and TRAP staining.

MTT assay

We obtained and cultured bone marrow-derived macrophages (BMMs) using the methods described previously [28]. Briefly, BMMs were obtained from the tibias and femurs and cultured in α-MEM supplemented with M-CSF (30 ng/mL). Cells were seeded in 96-well plates and incubated in complete medium containing various concentrations of erianin (0, 62.5, 125, and 250 nM) for 24, 48, or 72 h. We then performed a cell viability assay to assess the cytotoxic effects of erianin. Cell viability was quantified as a percentage of untreated cells.

Osteoclast differentiation and bone absorption assays

The BMMs were harvested and incubated on normal or hydroxyapatite-coated 96-well plates. Next, BMMs were cultured in α-MEM supplemented with 10% FBS, 30 ng/mL M-CSF, 50 ng/mL RANKL, or 5% 231 Conditional Medium (231 CM) and 2.5 ng/mL RANKL. The cells were treated with various concentrations of erianin (0, 62.5, 125, and 250 nM) until osteoclastogenesis, to assess its effects. After osteoclast formation, we performed a TRAP assay using a TRAP staining kit (Sigma) following the manufacturer's instructions. We then conducted a bone absorption assay by observing the resulting resorption pits using an Olympus IX71 light microscope and imaging software.

NF-κB luciferase reporter assay

The NF-κB-luc-RAW264.7 cells were cultured in plates using established methods [29]. The cells were pre-treated for 30 min with different working concentrations erianin (0, 31.75, 62.5, 125 and 250 nM) and BAY (1 μM), and were challenged under LPS for 6 h. A luciferase assay system was subsequently used to detect NF-κB luciferase activity.

Quantitative PCR and western blot assays

The BMMs were cultured in 12-well plates, and varying concentrations of erianin (0, 125, and 250 nM) were added to the culture medium for 72 h. An RNA isolation kit (Promega) was used to extract total RNA, which was then reverse-transcribed to cDNA using the PrimeScript RT reagent kit (Promega), and a qPCR Master Mix (Promega) was used to perform real-time PCR following the manufacturer's instructions. The primer sequences were as follows: c-Fos: forward 5′- TTG AGC GAT CAT CCC GGT C-3′, reverse 5′- GCG TGA GTC CAT ACT GGC AAG −3′; Trap, forward 5′- μCAC TCC CAC CCT GAG ATT TGT −3′, reverse 5′- CAT CGT CTG CAC GGT TCT G −3′; Nfatc1, forward 5′- GAC CCG GAG TTC GAC TTC G −3′, reverse 5′- TGA CAC TAG GGG ACA CAT AAC TG −3′; Gapdh, forward 5′- AGG TCG GTG TGA ACG GAT TTG −3′, reverse 5′- TGT AGA CCA TGT AGT TGA GGT CA −3′; Integrin β3, forward 5′- TGA CAT CGA GCA GGT GAA AG −3′, reverse 5′- GAG TAG CAA GGC CAA TGA GC −3′; c-Src, forward 5′- TTC CCT TCT GCA AAG GAG ATG T −3′, reverse 5′- ACC AGG GCA TAA GGC TGA GT −3′; Mmp9, forward 5′- GCG TCG TGA TCC CCA CTT AC −3′, reverse 5′- CAG GCC GAA TAG GAG CGT C −3′; Ctsk, forward 5′- GTT ACT CCA GTC AAG AAC CAG G −3′, reverse 5′- TCT GCT GCA CGT ATT GGA AGG −3′. Western blot assays of NFATc1, MMP9, INTEGRIN β3, SRC and p-SRC were performed according to the methods previously described [28] and the protein expression levels were analysed using ImageJ.

Cellular thermal shift assay

Cell lysates of BMMs were prepared, and the cell lysate supernatant was incubated with 100 μM erianin or DMSO (vehicle control) at 37 °C for 1 h. Subsequently, the lysates were divided into 100 μL aliquots and subjected to different temperature conditions (room temperature, 50, 60, 70, 80, 90, 100 °C). For the cellular thermal shift assay (CETSA), the heating process of the lysates was carried out for 3 min using a thermal cycler, followed by 5 min of cooling and then centrifuged at 4 °C to separate the soluble fractions. The resulting supernatants were mixed with loading buffer, boiled at 100 °C for 5 min, and separated by SDS-PAGE in 10% gels, to detect NFATc1 and SRC protein expression.

Knockdown of Nfatc1 and Src to examine the effects of erianin on osteoclast formation

Lipofectamine RNAIMAX and siRNA-transfected BMMs were used according to manufacturer’s instructions. The RNA oligo sequences used for transfection were as follows: Nfatc1-siRNA (sense: 5′-GUC AGU GUG ACC GAA GAU A(dT)(dT)-3′; antisense: 5′-UAU CUU CGG UCA CAC UGA C(dT)(dT)-3′), and Src-siRNA (sense: 5′-GGG AGA ACC UGG UGU GCA A(dT)(dT)-3′; antisense: 5′-UUG CAC ACC AGG UUC UCC C(dT)(dT)-3′). Transfection efficiency was analysed two days post-transfection. The BMMs were seeded on plates and transfected with siRNAs for 24 h. The plate was supplemented with RANKL and erianin (0 and 250 nM) for 4–5 days to perform TRAP staining.

GEPIA database analysis

Using the survival plot module of the Gene Expression Profiling Interactive Analysis (GEPIA) online database (https://gepia.cancer-pku.cn), we investigated the relationship between NFATc1 and RANKL gene expression, and the survival of patients with malignant tumours with a high risk of bone metastasis, such as breast and lung cancer.

Validation of erianin on osteoclastogenesis in patients with breast cancer with bone metastasis

Erianin treatment was clinically validated using a peripheral blood osteoclastogenesis system in patients with breast cancer with bone metastasis. We isolated peripheral blood mononuclear cells (PBMCs) from patients using Ficoll-Hypaque density-gradient centrifugation (DAKEWE). Subsequently, CD14⁺ monocytes were further separated from the PBMCs using Human Mono Iso Kit (Stemcell). Then 4 × 10⁴ CD14⁺ monocytes were distributed in normal or hydroxyapatite-coated 96-well plates, and cultured in media supplemented with M-CSF (30 ng/mL), RANKL (25 ng/mL), and varying concentrations of erianin (0, 125, and 250 nM). After two weeks, the cells in the plates were stained for TRAP or subjected to bone resorption assays.

Cellular immunofluorescent staining

Osteoclast precursors (BMMs or CD14⁺ monocytes) were cultured in 96-well plates, as described above. Fluorescent labelling was performed via using an NFATc1-specific mAb and a fluorescent secondary mAb, rhodamine-phalloidin, or calcium ion probe to determine NFATc1 nuclear transcription, F-actin ring organization, or Ca²⁺ oscillation, which were imaged using an ImageXpress Micro Confocal (MD, USA).

Statistical analysis

All statistical tests were conducted in GraphPad Prism (v. 8.0), and data in graphs are presented as mean ± SD. The Dunnett's multiple comparison post-hoc test was used to assess differences among various groups. A P value of less than 0.05 was selected as indicative of a statistically significant difference.

Results

Erianin ameliorates breast cancer cell-induced bone loss in vivo

We did not observe significant differences in body weight or other adverse events between the 2 and 20 mg/kg/day erianin-treated groups and other groups (Fig. 1C), suggesting that erianin did not exert any observable toxic effects during the course of treatment. The micro-CT results revealed that treatment with 2 and 20 mg/kg/day of erianin, effectively ameliorated trabecular bone loss induced by breast cancer (Fig. 1D). In agreement with this, erianin effectively rescued the breast cancer-induced reduction in bone mineral density (BMD), trabecular number (Tb.N), bone volume fration (BV/TV), connectivity density (Conn.D), and trabecular thickness (Tb.Tn) and increased bone parameters such as trabecular separation (Tb.Sp), structure model index (SMI), and bone surface fraction (BS/BV) (Fig. 1E–F). Moreover, H&E and TRAP staining revealed that treatment with erianin ameliorated bone destruction (Fig. 1G), accompanied by a reduced number of TRAP-positive cells, and a decrease in the ratios of osteoclast number to bone surface (N.Oc/BS), and osteoclast-covered surface area to bone surface (Oc.S/BS) (Fig. 1H). Taken together, our findings showed that erianin inhibited breast cancer cell-induced bone destruction in vivo.

Erianin attenuates breast cancer 231 CM-induced osteoclastogenesis and osteoclast-related gene expression, including Integrin β3/Mmp9

Considering the effect of erianin on breast cancer-induced bone destruction in vivo, we investigated whether erianin influenced breast cancer-induced osteoclast formation in vitro. Our results demonstrated that erianin at low concentrations ranging from 62.5 to 250 nM exerted an inhibitory effect on 231 CM-induced osteoclastogenesis, bone resorption (Fig. 2A–B) and F-actin ring generation (Fig. 2C) without any cytotoxicity (Fig. S1A). Next, we observed that 125, 250 nM erianin inhibited the expression of 231 CM-dependent osteoclast-related genes, such as Trap, Ctsk, Integrin β3, and Mmp9 (Fig. 2D). Moreover, erianin also effectively suppressed 231 CM-induced protein expression of INTEGRIN β3 and MMP9 (Fig. 2E–F). Overall, these results suggest that erianin attenuates osteoclastogenesis and the expression of osteoclast-related genes, such as Integrin β3 and Mmp9, induced by breast cancer.

Fig. 2.

Erianin inhibits breast cancer CM-induced osteoclastogenesis and expression of osteoclast-related genes including Integrin β3/Mmp9 (A) Representative images from TRAP and bone resorption pit assays for examining 231 CM-induced osteoclast formation. (B) Analysis of TRAP⁺ MNCs (nuclei > 3). The number and resorption area on hydroxyapatite-coated surface were counted (n = 5). (C) F-actin and nuclei (blue) images were visualized with fluorescence microscopy in 231 CM-induced osteoclasts with or without erianin treatment. (D) Gene expression of Trap, Ctsk, Integrin β3 and Mmp9 induced by 231 CM with different concentrations of erianin (n = 3). (E–F) Representative western blot images and accompanying analysis of β-Actin, INTEGRIN β3 and MMP9 protein expression induced by RANKL with or without erianin treatment (n = 3). Scale bar = 50 μm. The data are presented as mean ± SD (^#### p < 0.0001 vs control group; * p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001 vs 231 CM group). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Erianin inhibits the 231 CM-induced upregulation of SRC and NFATc1

Here, we explored the underlying mechanisms of action of erianin in breast cancer-induced osteoclastogenesis. The SRC kinase phosphorylation enhances Ca²⁺ oscillation and ultimately promotes the nuclear translocation of NFATc1 and downstream signalling [10], [11]. Our results demonstrated that 125 and 250 nM of erianin treatment inhibited Ca²⁺ oscillation that was activated by 231 CM (Fig. 3A–B), as well as downregulated the 231 CM-induced c-Src and Nfatc1 gene expression (Fig. 3C). At the post-transcriptional level, erianin also significantly reduced the propagation of the 231 CM signal in promoting SRC kinase phosphorylation and NFATc1 protein expression and nuclear translocation (Fig. 3D–H). Altogether, these findings provided evidence that erianin inhibits the breast cancer-induced SRC-NFATc1 pathway in breast cancer-induced pathological osteoclastogenesis.

Fig. 3.

Erianin inhibits the 231 CM-dependent upregulation of SRC-NFATc1 signalling (A–B) Representative view and quantitative analyses of Ca²⁺ oscillations induced by 231 CM (n = 3). (C) Real-time PCR quantification of c-Src and Nfatc1 gene expression following stimulation by 231 CM, with or without erianin treatment (n = 3). (D–E) Representative western blot images and band quantification of SRC, p-SRC, NFATc1 and β-Actin after induction by 231 CM, with different concentrations of erianin (n = 3). (F) Nuclear translocation of NFATc1 triggered by 231 CM, was detected by fluorescence imaging. (G–H) Representative western blot images and band quantification of NFATc1 expression in the nuclear fraction of RAW264.7 cells after incubation with 231 CM, with or without erianin treatment (n = 3). Scale bar = 50 μm. Data are presented as mean ± SD (^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 vs control group; * p < 0.05, ^** p < 0.01, ^*** p < 0.001 vs 231 CM group).

Erianin suppresses RANKL-induced osteoclastogenesis and the SRC-NFATc1-INTEGRIN β3 signal pathway

Breast cancer activates RANKL-dependent and-independent osteoclast formation [5]; therefore, we investigated the influence of erianin on RANKL-induced osteoclastogenesis. As depicted in Fig. 4A–B, erianin at concentrations of 62.5–250 nM inhibited osteoclastogenesis and bone resorption induced by RANKL, while fluorescence imaging revealed that erianin suppressed RANKL-induced F-actin ring formation (Fig. 4C). Moreover, based on the qPCR and western-blot assay results, both 125 and 250 nM of erianin effectively attenuated Trap, Ctsk, Integrin β3, and Mmp9 gene transcription (Fig. 4D), as well as the expression of the INTEGRIN β3, and MMP9 proteins (Fig. 4E–F), following RANKL-dependent osteoclastogenesis. Collectively, these findings demonstrate that erianin at concentrations of 125 and 250 nM inhibit RANKL-induced osteoclastogenesis and the expression of osteoclast-related genes.

Fig. 4.

Erianin suppresses RANKL-induced osteoclastogenesis and the SRC-NFATc1-INTEGRIN β3 signalling pathway (A) Representative images of TRAP and bone resorption pit assays. (B) Analysis of TRAP-positive multinucleated (nuclei > 3) cells. The number and resorption area on hydroxyapatite-coated surface induced by RANKL were quantified (n = 5). (C) F-actin and nuclei (blue) images were visualized by fluorescence imaging in RANKL-induced osteoclasts treated with varying concentrations of erianin. (D) Gene expression of Trap, Ctsk, Integrin β3, and Mmp9 induced by RANKL with or without erianin treatment, as assessed by real-time PCR (n = 3). (E–F) Representative western blot images and band quantification of INTEGRIN β3, MMP9 and β-Actin expression, following stimulation by RANKL, with or without erianin treatment (n = 3). (G–H) Representative view and quantitative analysis of Ca²⁺ oscillations induced by RANKL, with different concentrations of erianin (n = 3). (I) Gene expression of c-Src after RANKL induction, with or without erianin, detected by real-time PCR (n = 3). (J–K) Representative western blot images and band quantification of SRC, and p-SRC following RANKL stimulation, with or without erianin treatment (n = 3). (L) Representative fluorescence microscopy images of NFATc1 nuclear translocation induced by RANKL. (M−N) Representative western blot images and band quantification of NFATc1 expression in the nuclear fraction of RAW264.7 cells following RANKL induction, with or without erianin treatment (n = 3). Scale bar = 50 μm. The data are presented as mean ± SD (^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 vs control group; * p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001 vs RANKL group). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

In terms of the underlying mechanisms, fluorescence staining indicated that 125 and 250 nM of erianin suppressed Ca²⁺ oscillations triggered by RANKL (Fig. 4G–H). Erianin effectively inhibited the RANKL-induced upregulation of c-Src mRNA and phosphorylation of the SRC kinase (Fig. 4I–K). Furthermore, erianin suppressed NFATc1 protein expression in the nucleus and NFATc1 nuclear translocation under RANKL-induced osteoclastogenesis, as indicated by a fluorescence staining assay (Fig. 2L–N). However, the nuclear transcription of NF-κB and the gene expression of c-Fos remained unaffected by erianin at concentrations ranging from 31.25 to 250 nM during osteoclast formation (Fig. S1B–C). These findings highlight that SRC-NFATc1, rather than NF-κB-c-Fos, is the effector pathway through which erianin suppresses RANKL-induced osteoclastogenesis and expression of osteoclast-related genes, such as Integrin β3/Mmp9.

Erianin inhibits osteoclast formation by interaction with NFATc1 but not SRC

To investigate the potential drug targets of erianin during osteoclastogenesis, we performed CETSA and siRNA knockdown assays. The CETSA results revealed that erianin treatment did not confer protection to the SRC protein against thermal denaturation (Fig. 5A–B). Nevertheless, erianin significantly protected the thermal denaturation of NFATc1 protein at various temperatures, resulting in a marked shift to the right of the associated melting curves (Fig. 5C–D). Therefore, the CETSA results suggest that erianin directly interacts with NFATc1 but not SRC, during osteoclastogenesis. Next, we used a specific siRNA to knock down Nfatc1 and Src gene expression in BMMs, to evaluate the effects of erianin on osteoclastogenesis using the TRAP assay (Fig. 5E–I). Importantly, we observed that while Src-siRNA knockdown did not prevent erianin at 250 nM from suppressing osteoclastogenesis (Fig. 5F–G), this effect was abrogated after Nfatc1 knockdown (Fig. 5H–I). These results suggest that erianin directly interacts with NFATc1 instead of SRC, thereby inhibiting osteoclast formation. Moreover, the suppressive effects of 250 nM erianin on F-actin ring formation and NFATc1 nuclear translocation, were reduced after silencing Nfatc1 (Fig. 5J–L). In summary, these findings showcase that erianin directly interacts with NFATc1 and inhibits its function and osteoclastogenesis.

Fig. 5.

Erianin inhibits osteoclast formation by interacting with NFATc1 but not SRC (A) Representative western blot images of SRC protein levels with or without erianin treatment, assessed after incubation for 5 min at different temperature points (50, 60, 70, 80, 90, and 100 °C). (B) The corresponding CETSA melt curves of SRC (n = 3). (C) Representative western blot images of NFATc1 protein levels, with or without erianin treatment, assessed after incubation at varying temperature points. (D) The corresponding CETSA melt curves of NFATc1 (n = 3). (E) Real-time PCR quantification of Src and Nfatc1 gene expression following siRNA-mediated knockdown of Src or Nfatc1, respectively (n = 3). (F) Representative images of osteoclasts transfected with Src-siRNA or NC-siRNA, with or without erianin treatment. (G) Quantitative analysis of TRAP⁺ MNCs (nuclei > 3, n = 3). (H) Representative images of osteoclast transfected with Nfatc1-siRNA or NC-siRNA with or without erianin treatment. (I) Quantitative analysis of TRAP⁺ MNCs (nuclei > 3, n = 3). (J–K) Fluorescence microscopy imaging for monitoring the nuclear translocation NFATc1 in cells transfected with Nfatc1-siRNA or NC-siRNA, with or without erianin treatment (n = 3). (L) Representative images of osteoclasts’ F-actin ring formation, in cells transfected with Nfatc1-siRNA or NC-siRNA, with or without erianin treatment. Scale bar = 50 μm. Data are presented as mean ± SD (^#### p < 0.0001 vs. control group; * p < 0.05, ^** p < 0.01, ^*** p < 0.001).

Erianin inhibits excessive osteoclastogenesis in patients with breast cancer and bone metastases

By querying the GEPIA database, we conducted a survival analysis of over 1400 patients with cancer with a high risk of bone metastasis. We observed that higher NFATc1 gene expression was inversely proportional to the survival time of these patients (Fig. S2A), which did not occur in RANKL gene expression (Fig. S2B). We tested the clinical effects of the NFATc1 inhibitor erianin using a peripheral blood osteoclastogenesis system in patients with breast cancer with bone metastasis, as shown in Fig. 6A. The TRAP assay showed that treatment with 62.5, 125, and 250 nM erianin effectively reduced the formation and bone resorption of osteoclasts in patients with breast cancer (Fig. 6B–E) in a concentration-dependent manner. Notably, nearly no osteoclasts were observed after treatment with 250 nM erianin. Furthermore, the NFATc1 nuclear translocation and F-actin ring formation in patients with breast cancer, were inhibited by 125 and 250 nM erianin (Fig. 6F–H). These findings further corroborated that erianin acts an NFATc1 inhibitor, and reduces osteoclastogenesis in patients with breast cancer with bone metastases.

Fig. 6.

Erianin inhibits excessive osteoclastogenesis in patients with breast cancer and bone metastases (A) The specific process of osteoclastogenesis in patients with breast cancer with associated bone metastases in vitro. (B) Representative images of osteoclasts from CD14⁺ monocytes with different concentrations of erianin. (C) TRAP⁺ MNCs (nuclei > 3) were counted as osteoclasts (n = 3). (D) Images of bone resorption pit assays, depicting osteoclastogenesis from CD14⁺ monocytes with different concentrations of erianin. (E) Quantitative analysis of the resorption area on the hydroxyapatite-coated surface (n = 3). (F–G) Fluorescence microscopy imaging for monitoring RANKL-induced nuclear translocation NFATc1, with or without erianin treatment (n = 3). (H) Fluorescence microscopy images of the F-actin ring formation in osteoclasts incubated with different concentrations of erianin. Scale bar = 50 μm. The data are presented as mean ± SD (* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^**** p < 0.0001 vs RANKL group).

Overall, our study demonstrated that erianin is a novel NFATc1 inhibitor that directly interacts with NFATc1 but not SRC, and attenuates SRC-INTEGRIN β3-MMP9-dependent signalling, which consequently suppresses breast cancer-induced excessive osteoclastogenesis and bone loss in both mouse models and human patients.

Discussion

In cases of bone metastasis in breast cancer, the occurrence of SREs significantly affects the health status and expected lifespan of the patients. Osteoclast inhibition is widely recognized as an effective strategy to mitigate breast cancer-induced bone destruction and other SREs [2], [3], [4], [5], [6], [30]. The currently used drugs in clinic, namely zoledronic acid and denosumab, suppress the occurrence of SREs [18], [19]; however, their use may also lead to jawbone necrosis, resulting in rebound effects [31]. Furthermore, triple-negative breast cancer exhibits low RANKL secretion, which limits the effectiveness of RANKL monoclonal antibodies and other medications [32]. Therefore, there is an urgent need for novel therapeutic agents that can effectively inhibit SREs in patients with breast cancer with bone metastasis. Our findings show that erianin acts as a novel NFATc1 inhibitor to suppress breast cancer-induced osteoclastogenesis and bone destruction and may be a potential treatment for breast cancer-induced SREs.

First, we observed that erianin effectively mitigated breast cancer cell-induced mouse bone destruction and osteoclastogenesis. Bisphosphonate induced osteosclerosis in the mouse model of bone destruction [33]. While our results showed that erianin treatment only reversed the pathological bone loss in vivo. Erianin significantly inhibited osteoclastogenesis, F-actin ring formation, and osteoclast-related gene expression induced by 231 CM and RANKL, at a nM concentration in vitro. This indicates that erianin, a novel osteoclast inhibitor, effectively ameliorates breast cancer cell-induced bone loss via reducing osteoclastogenesis.

Next, we explored the underlying mechanisms of action of erianin in breast cancer-induced osteoclastogenesis. Breast cancer promotes osteoclastogenesis through RANKL-dependent and-independent pathways [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]. During RANKL-induced osteoclastogenesis, the phosphorylation of SRC enhances Ca²⁺ oscillations and ultimately promotes the nuclear translocation of NFATc1 [10], [11]. The NFATc1 is a key regulator of osteoclast formation [34], and induces the expression of crucial genes, including Integrin β3, Mmp9, Trap, Ctsk, ultimately leading to continuous activation of upstream and downstream signals and osteoclastogenesis [12]. The CM of cultured breast cancer cells contains IL-8 that induces RANKL-independent osteoclastogenesis [27]. We demonstrated that the breast cancer-derived 231 CM induced osteoclastogenesis and promoted the upregulation of NFATc1 expression and its nuclear translocation [27], [35]. Here, we showed that erianin inhibited the upregulation of Ca²⁺ oscillations, together with the SRC-NFATc1 and INTEGRIN β3-MMP9 signalling pathways during RANKL and breast cancer CM-induced osteoclast formation. These results indicate that erianin suppresses SRC and NFATc1 signaling to inhibit osteoclastogenesis and rescues breast cancer-induced bone loss.

We then investigated the drug target of erianin on osteoclast formation. We observed that erianin interacted with NFATc1 but not with SRC. Moreover, the knockdown of Nfatc1, but not Src, abrogated the inhibitory effects of erianin on osteoclast formation. In addition, knockdown of Nfatc1 blocked the effects of erianin on F-actin ring formation and NFATc1′s nuclear translocation during osteoclastogenesis. This suggests that erianin attenuates osteoclastogenesis by directly interacting with NFATc1 but not with SRC, leading to the inhibition of NFATc1′s nuclear translocation and the up-gulation of osteoclastogenesis.

Targeting NFATc1 signalling has been shown to have positive effects in many inflammatory diseases. For example, cyclosporine, a clinical drug for the treatment of multiple diseases, such as during organ transplantation [36], rheumatoid arthritis [37], ulcerative colitis [38], lupus nephritis [39] and psoriasis [40], inhibits NFATs signalling by targeting calcineurin. In addition, inhibitors that directly bind to NFATs, such as VIVIT, have also demonstrated therapeutic effects in disease models associated with renal fibrosis [41], osteoporosis [42], cardiovascular disorders [43], and allergic airway inflammation [44], and exhibit fewer side effects than cyclosporine [45]. Notably, VIVIT inhibited titanium particle-induced osteoclastogenesis [46] and excessive osteoclast activation in patients with rheumatoid arthritis [47]. A reduced expression of NFATc1 also reportedly ameliorates bone loss and osteoclast differentiation in an osteoporosis mouse model [34]. Our own findings highlight that erianin, which directly interacts with NFATc1, efficiently alleviated breast cancer-induced osteoclastogenesis and bone destruction in mice both in vitro and in vivo.

We also validated the effects of erianin on excessive osteoclastogenesis in patients with breast cancer. Our study revealed that the formation of osteoclasts in CD14⁺ monocytes from patients with breast cancer was suppressed by erianin treatment. Additionally, NFATc1 nuclear translocation was inhibited by erianin during osteoclastogenesis in monocytes of patients with breast cancer. The anti-RANKL antibody, denosumab, decreases the occurrence of SREs but does not affect breast cancer progression [21]. This is consistent with our analysis, indicating that high levels of RANKL expression did not affect the survival of patients with bone metastasis. Interestingly, our analysis identified a positive correlation between lower NFATc1 expression and longer survival in patients with bone metastasis. The novel NFATc1 inhibitor erianin reduced bone loss induced by breast cancer and almost completely eradicated excessive osteoclast formation of CD14⁺ cells in patients with breast cancer at nM concentrations. Moreover, erianin delays the progression of breast cancer [23], [48], a benefit that is not observed with bisphosphonate or denosumab therapy. Collectively, our findings suggest that erianin serves as a novel NFATc1 inhibitor to mitigate pathological osteoclastogenesis and prevent disease progression in patients with breast cancer.

Conclusions

In summary, our study demonstrated that erianin is a novel NFATc1 inhibitor that interacts with NFATc1 and efficiently mitigates breast cancer-induced osteoclastogenesis and bone destruction in vitro and in vivo. Moreover, 62.5–250 nM erianin inhibited excessive osteoclastogenesis of CD14⁺ monocytes in patients with breast cancer. These findings provide valuable insights into the treatment of SREs-induced pathological bone destruction and open new avenues for future applications of NFATc1 inhibitors.

Compliance with ethics requirements

All animal experiments were approved by the Institutional Animal Care and Use Committee of the Southern Medical University (SMUL202310005). All patients with breast cancer with bone metastasis were recruited from the Breast Surgery Department of The First Clinical School of Medicine Hospital of Jinan University, and the study was approved by the Medical Ethics Committee of The First Clinical School of Medicine Hospital of Jinan University (KY-2020–079).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

The following are the Supplementary data to this article: