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. 2023 Apr 29;46(5):1445–1456. doi: 10.1007/s13402-023-00822-9

Repurposing ketotifen as a therapeutic strategy for neuroendocrine prostate cancer by targeting the IL-6/STAT3 pathway

Yiyi Ji 1,#, Bo Liu 1,#, Lei Chen 1,#, Ang Li 1, Kai Shen 1, Ruopeng Su 1, Weiwei Zhang 1, Yinjie Zhu 1,✉, Qi Wang 1,2,✉, Wei Xue 1,✉
PMCID: PMC12974647  PMID: 37120492

Abstract

Purpose

Neuroendocrine prostate cancer (NEPC), a highly aggressive subtype of prostate cancer displaying resistance to hormone therapy, presents a poor prognosis and limited therapeutic options. Here, we aimed to find novel medication therapies for NEPC and explore the underlying mechanism.

Methods

A high-throughput drug screening utilizing an FDA-approved drug library was performed and ketotifen, an antihistamine agent, was identified as a potential therapeutic candidate for NEPC. The whole-transcriptome sequencing analysis was conducted to explore mechanism of ketotifen inhibitory in NEPC. Multiple cell biology and biochemistry experiments were performed to confirm the inhibitory effect of ketotifen in vitro. A spontaneous NEPC mice model (PBCre4:Ptenf/f;Trp53f/f;Rb1f/f) was used to reveal the inhibitory effect of ketotifen in vivo.

Results

Our in vitro experiments demonstrated that ketotifen effectively suppressed neuroendocrine differentiation, reduced cell viability, and reversed the lineage switch via targeting the IL-6/STAT3 pathway. Our in vivo results showed that ketotifen significantly prolonged overall survival and reduced the risk of distant metastases in NEPC mice model.

Conclusion

Our findings repurpose ketotifen for antitumor applications and endorse its clinical development for NEPC therapy, offering a novel and promising therapeutic strategy for this formidable cancer subtype.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13402-023-00822-9.

Keywords: NEPC, Ketotifen, Lineage switch, Drug repurposing, High-throughput drug screening, IL-6/STAT3 pathway

Introduction

Neuroendocrine prostate cancer (NEPC) is a highly aggressive subtype of prostate cancer, characterized by significant heterogeneity and poor differentiation. It is marked by the expression of neuroendocrine markers and the loss of androgen receptor (AR)-related pathways, often arising as a result of androgen deprivation therapy [1, 2]. The incidence of treatment-induced NEPC is escalating due to the increasing clinical applications of AR-targeting agents, such as enzalutamide and abiraterone [3]. NEPC bears clinical features distinct from usual prostate adenocarcinoma, including markedly shorter median overall survival, a higher incidence of lethal visceral metastases, and lower sensitivity to androgen deprivation therapy or docetaxel-based chemotherapy [4].

Previous research has shown that the neuroendocrine differentiation of prostate cancer is associated with a variety of genetic alterations. Specifically, the collective loss of RB1 and PTEN fosters lineage plasticity and metastasis, whereas the combined loss of RB1 and TP53 results in resistance to androgen deprivation therapy [5-7]. Specially, deletion of Pten, Rb1, and Trp53 in prostate epithelial cells has been proven to induce the development of spontaneous neuroendocrine prostate cancer in mice model (PBCre4:Ptenf/f;Trp53f/f;Rb1f/f) [8]. In addition, the activation of the IL-6-signal transducer and activator of transcription 3 (STAT3) signaling pathway triggers lineage switch and is essential for the stem-like properties [9, 10], characteristics of epithelial-mesenchymal transition (EMT) [11, 12], resistance for AR-targeted therapy [13], and appearance of neuroendocrine phenotype [14, 15]. Furthermore, the co-expression of MYCN and activated AKT1 is sufficient to generate NEPC cell line LASCPC-01 from normal prostate epithelia [16].

Lineage plasticity pertains to the transformation of luminal prostate epithelial cells into a lineage-plastic state, resulting in a stem cell-like, neuroendocrine-like, and AR-targeted therapy-resistance cells [17, 18]. This plasticity is normally attributed to the amplification of key transcription factors (such as MYCN and SOX2) [19, 20] as well as the activation of IL-6/STAT3 signaling pathways [9]. Identifying druggable targets responsible for driving neuroendocrine differentiation and lineage plasticity in NEPC is critical to developing effective therapies. While platinum-based chemotherapy remains the first choice for NEPC patients, response rates and survival outcomes are generally poor [2, 21]. Indeed, new approaches targeting drivers of NEPC are emerging, including (1) Alisertib, an Aurora kinase A (AURKA) inhibitor targeting the interaction of MYCN and AURKA [22]; (2) rocalpituzumab tesirine, an agent targeting DLL3 [23]; (3) avelumab, an PDL1 inhibitor [24]. However, these approaches failed to meet the primary endpoint in phase II clinical trials, and other novel drugs are still undergoing evaluation. As such, new therapeutic strategies for NEPC are still required.

In our study, we performed a high-throughput screening and identified ketotifen, an FDA-approved antihistamine, as a potential therapy for NEPC. Our results suggested that ketotifen effectively reduced cell viability, suppressed neuroendocrine differentiation, and inhibited lineage switch by targeting the IL-6/STAT3 pathway in NEPC cells. Our in vivo results showed that ketotifen significantly prolonged overall survival and reduced the risk of distant metastases in NEPC mice model. Therefore, our findings suggest that ketotifen has the potential to be repurposed as a novel antagonist for the STAT3 pathway and a promising therapeutic strategy for NEPC.

Methods

Drug screening

Using the Cell Explorer High Throughput Screening Workstation (PerkinElmer®), we screened a total of 1113 FDA-approved drugs from the drug libraries against the LASCPC-01 cell line. Prior to the screening, cells were seeded in the CulturPlate 384-well plate (PerkinElmer®) and exposed to individual compounds at three different concentrations: 0.4 μM, 1 μM, and 2 μM. The Multidrop Combi (Thermo Scientific®) was used for dispensing cell suspension or reagent. Cell viability was assessed by CellTiter-Glo Luminescent Assay (Promega®, G7572) following the manufacturer’s protocol after 72 h of incubation.

Cell line and Organoid culture

The human prostate cancer cell line LASCPC-01 and PC3 were purchased from the American Type Culture Collection (ATCC). HITES medium modified with hydrocortisone, human recombinant insulin, transferrin, estradiol, and estradiol was used to culture LASCPC-01 cells. And PC3 cells were cultured in RPMI-1640 medium (Gibco, C224005500). 10% fetal bovine serum (Gibco, A3161002C) and 100 U/mL penicillin–streptomycin (Thermo Fisher Scientific, 15140122) were added to all the tumor cell conditional medium. All the cells were cultured in a humidified incubator at 37℃ with 5% CO2. The drug reagent ketotifen was purchased from MedChemExpress (MCE, HY-B0157) and applied according to the recommended manufacturer’s protocol. Ketotifen was pre-mixed with complete culture medium in advance and diluted to working concentrations.

Murine prostate tumor tissues were collected, minced and digested in DMEM culture medium (Gibco, 11965092) containing 0.2 mg/mL collagenase (Gibco, 17100017), 0.1 mg/mL DNase I (STEMCELL Technologies, 07469), 1U/mL dispase (Sangon, 78,990-62-2) and 2% FBS (Gibco, A3161002C). The digestion was performed on a shaker for 1 h at 37 °C. We blocked tumor cells with CD16/32 antibody for 40 min and then stained them with EpCam antibody (BioLegend, 2%FBS in PBS) for 1 h on ice. Anti-Biotin Microbeads (Miltenyi Biotec) were added into the cell suspension for incubation at 4 °C. We used LS columns (Miltenyi Biotec) to isolate EpCam (+) tumor epithelial cells with the magnetic field produced by a magnetic cell sorting (MACS) separator. Then the purified tumor cells were cultured according to a previously reported method [25].

Real Time Quantitative PCR (RT-qPCR)

The method of TRIzol Reagent (Ambion, 15596026) was followed to prepare total RNA in prostate cancer cells. A NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific) was used to assesse the concentration and quality of total RNA. cDNA was synthesized with HiScript III RT SuperMix for qPCR (Vazyme, Q121-02) by using 2 μg of RNA. Then, RT-qPCR was carried out with ChamQ Universal SYBR qPCR Master Mix reagent (Vazyme). The 2−ΔΔCq method detected relative gene mRNA expression level and normalized it to the reference gene and indicated control group. Primers used in this study was indicated in supplementary Table S1.

Western blot analysis

RIPA lysis buffer with 1% PMSF and 1% Protease inhibitor cocktail added (Beyotime Biotechnology, ST506, P1112) was employed to extract the whole protein from cells. The BCA protein assay kit (Thermo Fisher Scientific, 23225) was used to assess the concentration of protein. A minimum of 10 μg protein samples were applied onto 10% SDS/PAGE gel. Prestained Protein Ladder (Thermo Fisher Scientific, 26616) served as protein markers. Subsequently, the proteins were transferred to 0.45 μM PVDF membranes after running program. Then, membranes were blocked with 5% non-fat milk (in TBST) for at least 1 h at room temperature. Afterwards, the membranes were incubated with the diluted primary antibodies at 4℃ overnight. Horseradish peroxidase-labeled goat anti-mouse or anti-rabbit IgG second antibody was incubated with the membranes for 1 h at room temperature. BeyoECL Plus enhanced chemiluminescence reagent (Beyotime Biotechnology, P0018M) was used to visualize the signals in membranes; and gel imaging analysis (Tanon) collected images. Antibodies used in this study was indicated in supplementary Table S2.

Celltiter-Glo

To evaluate the cell viability and estimate the IC50 of ketotifen in LASCPC-01 and prostate organoid, CellTiter-Glo Luminescence Assay (Promega®, G7570) was applied. Cells were seeded in a 96-well plate at 10,000 cells/well and treated with various concentrations of ketotifen. CellTiter-Glo Regent was used to lyse the cells after 48 h of incubation. The luminescence signal from ATP in viable cells was detected by a plate reader 10 min post-lysis.

Flow cytometry

The prostate cancer cells were cultured in a 10 cm dish with culture medium at a density of 5 × 106 LASCPC-01 cells per dish and subjected to the designated dose of ketotifen (dissolved in PBS) at 37 °C prior to collection. Subsequently, the LASCPC-01 cells were washed with PBS and fixed in 70% ethanol in PBS overnight at -20 °C. The fixed cells were then stained with DAPI (10 μg/mL) and treated with RNase A (100 ng/mL) for 30 min at 37 °C. Cell cycle analysis was performed using BD FACSCanto flow cytometry (BD Biosciences, v3.0).

Colony formation assay

The clonogenic activity of prostate cancer cells (PC3/Ctrl and PC3/MYCN) was assessed by colony forming assay. A total of 1000 cells per well were seeded in a 6-cm dish with conditioned medium and incubated for 7 days until single cell clones were developed. The cells were then washed with PBS three times, fixed with 4% paraformaldehyde for 20 min and stained with 0.5% crystal violet. The cell colonies were quantified using ImageJ software (v2.1.0).

Wound healing assay

The wound healing assay was performed as follows: PC3/MYCN cells were cultured in 6-well plates until they reached 80–90% confluence. A flat wound line was created using a 200-μL pipette tip. The plates were then washed with PBS to remove shed cells and the medium was replaced with serum-free RPMI-1640 to control cell proliferation. The cell scratch was photographed using a light microscope at 0 and 16 h. The wound healing rates were then determined using Image J software (v2.1.0).

Transwell assay

PC3/MYCN cells (5 × 105) were seeded onto 8-μm pore size inserts in 24-well plates with serum-free RPMI-1640. The lower chamber was filled with 700 μL of medium containing 10% FBS as a chemoattractant. After incubation for 12 h at 37 °C, non-migrated cells on the upper surface of the membrane were removed with a cotton swab. Migrated cells on the lower surface of the membrane were fixed, stained, and counted under a light microscope.

RNA sequencing

Total RNA was isolated from LASCPC-01 cells exposed to Ketotifen or PBS using TRIzol Reagent (Ambion, 15596026) based on the manufacturer’s protocol. The purity, quantification, and integrity were measured using NanoDrop 2000 spectrophotometer (Thermo Scientific) and Agilent 2100 Bioanalyzer (Agilent Technologies). Then we constructed libraries using VAHTS Universal V6 RNA-seq Library Prep Kit following the manufacturer’s instructions. Differential expression analysis was done using DESeq2. Hierarchical cluster analysis of the differentially expressed genes (DEG) was done using R-software (3.2.0) to display the gene expression pattern in different samples. Gene set enrichment analysis (GSEA) was done using GSEA software to examine the altered gene sets in the drug-treated group. Significance was defined as NES > 1.0, P value < 0.05, and FDR < 0.25.

Animal experiments

The ethics committee of Ren Ji Hospital approved all animal experiments in this study. Triple knocked-out (PBCre4:Ptenf/f;Trp53f/f;Rb1f/f) mice were used to construct NEPC models. They were castrated at 9 weeks with 1% isoflurane anesthesia on a heating pad and housed under standard conditions with free access to water and food. They were randomly assigned to two groups for subsequent experiments at 10 weeks of age. Intraperitoneal (i.p.) injections of Ketotifen at 5 mg/kg or a matching vehicle volume were administered every 2 days and their body weight was recorded each time. The NEPC mice were executed by anesthesia and rapidly dissected after 4 weeks of injections. Their prostates, lungs, livers, and lymph nodes were harvested. In addition, the above experiment was repeated until they were moribund to record their overall survival. Their body weight and viability were monitored, and the date of death was recorded in time.

Hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) analysis, and ELISA

Prostate tumor samples were collected from NEPC mice models treated with ketotifen or PBS. The samples were fixed with 4% paraformaldehyde and paraffin-embedded, then cut into 4 μm sections. H&E staining was done following standard protocols. For Immunohistochemistry analysis, different primary antibodies were applied to measure the protein levels of SYP, Ki67, Cleaved-Caspase-3, and p-STAT3 in the tumor tissue. After Diaminobenzidine Tertrahydrochloride (DAB) detection, all the sections were mounted with neutral gum. A light microscope was used to capture images of the sections, and ImageJ software (v2.1.0) was used to calculate the H-score of these images. Mouse IL-6 ELISA kit (#RK00008, ABclonal) was used to measure the IL-6 protein levels in tumor tissue from NEPC mice models treated with ketotifen or PBS for 4 weeks. Antibodies used in this study was indicated in supplementary Table S2.

Statistical analysis

Each experiment was performed at least three times independently and the mean and standard deviation (SD) of the results were reported. The ketotifen group was compared with the control group using Student’s t-test. All the data analysis was done with GraphPad Prism 9.0. P value < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Results

High-throughput drug library screening identifies that ketotifen inhibits the NEPC cell proliferation

To develop novel medication therapies for NEPC, we performed a drug screening using a NEPC cell line, LASCPC-01, employing a drug library including 1113 FDA-approved drugs. As depicted by the workflow schema of drug screening in Fig. 1A, candidates were tested over 72 h at the concentrations of 0.4 μM, 1 μM, and 2 μM. As shown in Fig. 1B, we selected 15 drugs based on the following criteria: 1) the ability to suppress LASCPC-01 cell proliferation (relative cell viability < 1) at 0.4 μM; 2) the significant inhibitory activity (relative cell activity < 0.5) at 2 μM; 3) the concentration-dependent inhibitory effects at the aforementioned three concentrations. Unlike one-concentration assays, this concentration-dependent monitoring allowed us to rule out unspecific drugs for treating NEPC cells. This approach identified several candidates with differential effects, including DNA synthesis inhibitors such as Mechlorethamine and Melphalan; FGFR inhibitor such as Erdafitinib, and Histamine H1 receptor inhibitors such as Ketotifen fumarate and Dimenhydrinate. Among the 15 hits obtained, we selected ketotifen due to its superior inhibitory effect, well-established safety profile, and widespread clinical use, aiming to expand the inhibitory effects on NEPC models and explore the detailed mechanism.

Fig. 1.

Fig. 1

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High-throughput drug library screening identifies that ketotifen inhibits the NEPC cell proliferation. A Schematic illustration of the whole experiment flow. B The effectiveness of each compound in inhibiting LASCPC-01 cells. FDA-approved drugs (blue), drugs suppress LASCPC-01 cell proliferation (relative cell viability < 1) at 0.4 μM (orange), (relative cell activity < 0.5) at 2 μM (red) were labelled. And table showing the cell viability of 15 candidates

Ketotifen effectively reduces NEPC viability and induces apoptosis

To explore the potential applications of ketotifen in other NEPC models, we utilized an organoid model derived from the tumor of NEPC transgenic mice (PBCre4:Ptenf/f;Trp53f/f;Rb1f/f). As shown in Fig. 2A, we found that ketotifen reduced both the viability and size of organoids in a concentration-dependent manner. Additionally, we performed cell cycle cytometry analysis in LASCPC-01 cells and observed a significant increase in the percentage of sub-G1 in ketotifen group, indicating that ketotifen treatment induced cell apoptosis (Fig. 2B). Western blot showed that the apoptotic cell death was a consequence of ketotifen application at 5 μM in a time-dependent manner (Fig. 2C).

Fig. 2.

Fig. 2

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Ketotifen effectively reduces NEPC viability and induces apoptosis. A The viability and size of organoid derived from PBCre4:Ptenf/f;Trp53f/f;Rb1f/f mice model tumor in a dose-dependent manner. Representative images were shown. Scale bar, 100 µm. B Cell cycle analysis of LASCPC-01 cells after 48 h treatment with PBS or 5 µM ketotifen (left). Quantification of the sub-G1 fraction was shown (right). C Western blot showing the induction of apoptosis markers after ketotifen treatment in LASCPC-01 cells in a time-dependent manner. D Bar plots showing the enrichment of significantly downregulated oncogenic traits after ketotifen treatment in LASCPC-01 cells. E Cell viability of PC3/Ctrl and PC3/MYCN cells treated with increasing concentration of ketotifen for 48 h. F Colony formation assay of PC3/Ctrl and PC3/MYCN cells treated with increasing concentration of ketotifen for 7 days

To gain further insight into the impact of ketotifen on NEPC, we conducted whole-transcriptome sequencing on LASCPC-01 cells with ketotifen or PBS treatment for 48 h. As shown in Fig. 2D, we revealed a significant enrichment of downregulated oncogenic traits after ketotifen treatment, providing further evidence of its inhibitory effects on LASCPC-01 cells. We overexpressed MYCN in PC3 cells to further evaluate specific inhibitory effects of ketotifen in NEPC models and observed that PC3/MYCN exhibited greater sensitivity to ketotifen in comparison with PC3/Ctrl cells, as evidenced by cytotoxicity assays and colony formation assays (Fig. 2E and 2F)

Ketotifen suppresses neuroendocrine phenotype and inhibits epithelial-mesenchymal transition in NEPC

Transcriptome sequencing analysis suggested that ketotifen treatment upregulated AR pathway and reduced the expression of neuroendocrine-related transcription factors (Fig. 3A), such as EZH2 and ONECUT2 [20, 26, 27]. Consistently, we observed ketotifen inhibited the expression of neuroendocrine markers in LASCPC-01(Fig. 3B and 3C). Following confirmation of the expression of MYCN and neuroendocrine markers (Fig. 3D and 3E), to further confirm the inhibitory effect of ketotifen in NEPC phenotype at the cellular level, we treated PC3/MYCN with ketotifen and observed the decreased expressions of neuroendocrine markers including NCAM1, CHGA, SYP, and NSE (Fig. 3F). As indicated in Fig. 3G, the pathway enrichment analysis suggested that EMT pathway was inhibited after ketotifen treatment. Consistently, ketotifen also decreased EMT-related genes as detected by qPCR (Fig. 3H). Subsequently, we validated that PC3/MYCN cells treated with ketotifen exhibited reduced migration compared with PC3/Ctrl (Fig. 3I). Also, wound healing assay revealed ketotifen reduced migration ability of PC3/MYCN compared with PC3/Ctrl (Fig. 3J). Together, our findings demonstrated that ketotifen inhibited cell proliferation, suppressed neuroendocrine phenotype, and inhibited EMT progression in NEPC in vitro.

Fig. 3.

Fig. 3

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Ketotifen suppresses neuroendocrine phenotype and inhibits epithelial-mesenchymal transition in NEPC. A Heatmap showing the transcriptome analysis of AR pathway and NE-related transcription factors (TFs) in LASCPC-01 cells treated with 5 µM ketotifen in comparison to the PBS treated cells. B Relative expression of NE-related markers reduced in LASCPC-01 cells treated with 5 µM ketotifen or PBS. C Western blot showing reduction of NE-related markers after ketotifen treatment in LASCPC-01 cells in a dose-dependent manner. D Western blot showing the overexpression of MYCN in PC3 cells. E Relative expression of NE-related markers increased in PC3/MYCN as compared to PC3/Ctrl cells. F Relative expression of NE-related markers reduced in PC3/MYCN cells treated with 2 or 5 µM ketotifen in comparison to PBS. G Heatmap showing the transcriptome analysis of EMT-related genes in LASCPC-01 cells treated with 5 µM ketotifen or PBS. H Relative expression of EMT-related markers reduced in PC3/MYCN cells treated with 2 or 5 µM ketotifen in comparison to PBS. I Transwell assay showing the reduced migration of PC3/MYCN cells in response to treatment with 5 µM ketotifen. J Wound healing assay of PC3/MYCN cells treated with 5 µM ketotifen or PBS. Scale bar, 100 μm

Ketotifen exerts its anti-neuroendocrine effects by inhibiting the IL-6/STAT3 pathway

Next, we conducted a gene set enrichment analysis (GSEA) and revealed a significantly reduced enrichment of IL-6/JAK/STAT3 signaling pathway after treatment of ketotifen (Fig. 4A). Heatmap of the transcriptome analysis also indicated that ketotifen inhibited the expression of various IL-6/JAK/STAT3 signature genes (Fig. 4B). Subsequently, we conducted western blot analysis to confirm the inhibitory effect of ketotifen on the IL-6/JAK/STAT3 pathway. As shown in Fig. 4C, the phosphorylation level of STAT3 and the expression of neuroendocrine markers were significantly decreased after ketotifen treatment. Previous studies reported that the lineage switch of prostate cancer cells from luminal to basal and stem-like cell types contributes to the acquisition of the neuroendocrine phenotype and aggressive variants [28, 29]. Our transcriptome analysis revealed the upregulation of luminal signature, as well as downregulation of basal and stem-like signature (Fig. 4D). Importantly, ketotifen treatment decreased basal marker genes (KRT5 and TP63) and stem-like marker genes (SOX2 and NANOG) (Fig. 4E), which further verified the inhibitory effects of ketotifen on lineage switch in NEPC models.

Fig. 4.

Fig. 4

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Ketotifen exerts its anti-neuroendocrine effects by inhibiting the IL-6/STAT3 pathway. A Gene Set Enrichment Analysis in LASCPC-01 cells showing IL-6/JAK/STAT3 pathways downregulated after ketotifen treatment. B Heatmap showing the transcriptome analysis of IL-6/JAK/STAT3 signature in LASCPC-01 cells treated with 5 µM ketotifen or PBS. C Western blot showing the levels of the IL-6/JAK/STAT3 related and NE-related pathways after ketotifen and IL-6 treatment in LASCPC-01 cells. D Heatmap showing the transcriptome analysis of luminal-related, basal-related, and stem-like in LASCPC-01 cells treated with 5 µM ketotifen in comparison to PBS. E Relative expression of basal-related and stem-related genes reduced in PC3/MYCN cells treated with 2 or 5 µM ketotifen in comparison to PBS

Furthermore, our transcriptome analysis suggested that ketotifen also inhibited several oncogenic traits, such as the NF-κB pathway and the PI3K/AKT/mTOR pathway (Fig. S1A and S1D). Consistently, we confirmed that ketotifen could inhibit NF-κB downstream signaling factors in mRNA level and suppress the phosphorylation level of AKT (Fig. S1B and S1C).

Ketotifen prolongs the overall survival of NEPC mice and suppresses the neuroendocrine differentiation of tumor.

To further investigate the effect of ketotifen in vivo, we castrated NEPC mice (PBCre4:Ptenf/f;Trp53f/f;Rb1f/f) when they are 9 weeks old. Then, we treated the pre-castrated mice with ketotifen or PBS every 2 days for 4 weeks (Fig. 5A). Interestingly, we observed ketotifen significantly prolonged the overall survival of NEPC mice (Fig. 5B). Our immunohistochemistry analysis confirmed that ketotifen significantly decreased the expression of Ki67 and SYP, while simultaneously increased the expression of Cleaved Caspase-3 (Fig. 5C). In addition, EdU incorporation assay revealed that ketotifen inhibited DNA replication and proliferation (Fig. 5D). Moreover, we observed the reduced number of visceral metastases in both liver and lung, indicating a suppression of migration ability of tumor cells (Fig. 5E and 5F). Importantly, we found that ketotifen reduced the IL-6 level and p-STAT3 in NEPC mice tumor tissue after 4 weeks ketotifen treatment (Fig. 5C and 5G), suggesting that ketotifen inhibited NEPC by downregulating the IL-6/STAT3 pathway. We also monitored the body weights of these mice and observed no significant differences between ketotifen group and PBS group (Fig. 5H), suggesting the safety of ketotifen. Together, ketotifen prolonged the overall survival of NEPC mice and inhibited tumor proliferation, neuroendocrine phenotype, and metastases in NEPC mouse model.

Fig. 5.

Fig. 5

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Ketotifen prolongs the overall survival of NEPC mice and suppresses the neuroendocrine differentiation of tumor. A Schematic illustration of the ketotifen treatment in NEPC mice. Mice were injected with ketotifen (5 mg/kg) or vehicle (i.p.). B The survival of NEPC mice treated with ketotifen or vehicle. C Representative images and quantification of SYP, Ki67, Cleaved Caspase-3 (CC3), and p-STAT3 staining of mice prostate tumor tissue. Scale bar, 50 μm. D Representative immunofluorescence staining of EdU in tumors from NEPC mice treated with ketotifen or vehicle. Scale bar, 50 μm. E Representative H&E staining images of mice tumor metastasis (liver and lung). Black arrows point to sites of tumor metastasis. Scale bar, 4 mm. F Table showing quantifications of tumor metastatic sites (liver, lung, and lymph node) in NEPC mice treated with ketotifen or vehicle. G ELISA assay showing the IL-6 concentration reduced in tumor samples from NEPC mice treated with ketotifen. H Body weight of mice treated with ketotifen or vehicle

Discussion

NEPC is a highly aggressive subtype of prostate cancer, with rare therapeutic options, poor prognosis, and resistance to hormone therapy. Currently, cisplatin-based chemotherapy is the primary first-line treatment for NEPC, but its clinical efficacy is unsatisfactory. Therefore, there is an urgent requirement for novel, potent, and safe therapies for NEPC patients. Importantly, repurposing FDA-approved drugs through high-throughput screening represents a valuable strategy to expedite drug development and reduce initial costs, as these candidates have already undergone clinical trials.

Originally, ketotifen is an antihistamine widely used in treating allergic conditions by blocking the action of histamine and stabilizing the mast cells through reducing the release of neurotransmitters. Interestingly, a prior study indicated that ketotifen could be a potential therapeutic strategy for neurofibromas in patients with NF1 by reducing the migration and growth of mast cells[30]. Additionally, ketotifen has been reported as a modulator of exosomes releasing from cancer cells, reducing their resistance to chemotherapy [31]. However, there is still a scarcity of research regarding both the utilization and the underlying mechanism of ketotifen in the treatment of cancer.

In our study, we performed a high-throughput screening utilizing an FDA-approved drug library and examined the potential of ketotifen as a therapeutic candidate for NEPC. Our findings demonstrated that ketotifen effectively inhibits neuroendocrine differentiation, curbs cell viability, and reverses the lineage switch of NEPC cell lines, including LASCPC-01, PC3/MYCN, and organoids derived from tumor of NEPC mice model.

Previous studies suggested that NF-κB family transcription factors play a role in the downstream signaling of Histamine 1 receptor (H1R) [32], and that blocking H1R with ketotifen lead to a decrease in TNF-α and NF-κB signaling pathway [33]. Moreover, various studies have reported that ketotifen treatment can significantly reduce the levels of TNF-α and IL-6 in serum of diabetic rats or liver with ischemia/reperfusion [34, 35]. Our results are consistent with these findings, showing that ketotifen significantly inhibit the NF-κB signaling and decrease the IL-6 level, thereby inhibiting the critical IL-6/STAT3 signaling pathway via suppressing both the endogenous and exogenous phosphorylation of STAT3 (Fig. 4D). This leads to a pronounced reduction in cell proliferation and disruption in lineage plasticity. The activation of IL-6/STAT3 signaling pathway has been shown to promote characteristics of EMT [11, 12], and our transcriptome analysis also suggested ketotifen inhibited EMT. Consistently, our transwell assay and wound healing assay revealed that ketotifen reduced the migration capacity of PC3/MYCN cells (Fig. 3F and 3G). Intriguingly, another antihistamine, Dimenhydrinate, also exhibits a potential inhibitory effect on LASCPC-01 cells, indicating the need for further investigation of histamine antagonist in NEPC therapy.

Since a number of recent studies have shown the pivotal role of the JAK/STAT pathway in enhancing the adaptability of prostate cancer cells [9], there are currently no FDA-approved drugs available for this oncogenic trait. Our in vivo experiments utilizing NEPC mice model showed that ketotifen significantly prolongs overall survival and reduces the risk of distant metastases. Overall, our findings provide a strong experimental basis for using ketotifen as a clinical treatment for NEPC. Although our study shows promise for ketotifen in NEPC therapy, we must acknowledge its current limitations. Firstly, further research is warranted to uncover the underlying molecular mechanisms of ketotifen and its effects on the JAK/STAT pathway. Secondly, it is imperative to evaluate the safety and efficacy of ketotifen in a broader range of preclinical and clinical models to establish its potential for therapeutic use.

In conclusion, our study underscores the great therapeutic potential of ketotifen in the treatment of NEPC by inhibiting the STAT3 signaling pathway, and provides new insights into the molecular mechanisms of ketotifen.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 632 KB) (632.3KB, docx)
Supplementary file2 (PDF 6832 KB) (6.7MB, pdf)

Abbreviations

NEPC

Neuroendocrine prostate cancer

FDA

Food and Drug Administration

IL-6

Interleukin 6

STAT3

Signal transducer and activator of transcription 3

JAK

Janus Kinase

AR

Androgen receptor

EMT

Epithelial-mesenchymal transition

PTEN

Phosphatase and tensin homolog

TP53

Tumor protein 53

RB1

Retinoblastomal 1

MYCN

V-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog

AKT1

AKT Serine/Threonine Kinase 1

PBS

Phosphate buffer saline

IHC

Immunohistochemistry

H&E

Hematoxylin-eosin

p-

Phosphorylated

Authors’ contributions

QW, and WX contributed to conception and design of the study. YJ, BL, and LC organized the database. YJ, RS, and KS performed the statistical analysis. YJ, BL, and LC wrote the first draft of the manuscript. AL and WZ wrote sections of the manuscript. QW, YZ, and WX obtained the funding. All authors contributed to manuscript revision, read, and approved the submitted version.

Funding

The work was supported by National Natural Science Foundation of China (No 82072847, 81772742, 82172868, 81972578), Ren Ji Hospital (PYI20-04, LYZXHXKT220845, 2020LYRJ-002, PNO-0106, RJKY18-02). The funders had no role in study design, data collection and analysis, decision to publish, or manuscript preparation.

Data availability

All pertinent data and materials can be obtained from the corresponding author upon a reasonable request. The RNAseq data of LASCPC-01 cells treated with ketotifen or PBS was uploaded to the Gene Expression Omnibus database GSE225007.

Declarations

Ethics approval

All mice experimental procedures were approved by the Research Ethics Committee of Ren Ji Hospital. The research was conducted in accordance with the Ethical Principles of Measures for Ethical Review of Biomedical Research Involving Human Beings and the Declaration of Helsinki.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Footnotes

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Yiyi Ji, Bo Liu and Lei Chen contributed equally to this work and share first authorship.

Contributor Information

Yinjie Zhu, Email: yinjiezhu@outlook.com.

Qi Wang, Email: wqi@sjtu.edu.cn.

Wei Xue, Email: uroxuewei@163.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (DOCX 632 KB) (632.3KB, docx)
Supplementary file2 (PDF 6832 KB) (6.7MB, pdf)

Data Availability Statement

All pertinent data and materials can be obtained from the corresponding author upon a reasonable request. The RNAseq data of LASCPC-01 cells treated with ketotifen or PBS was uploaded to the Gene Expression Omnibus database GSE225007.

Articles from Cellular Oncology are provided here courtesy of Springer

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