Blockade of neutral sphingomyelinase 2 exerts antitumor effect on metastatic castration resistant prostate cancer cells and promotes tumor regression when combined with Enzalutamide

Abstract

Prostate cancer (PCa) is the second leading cause of cancer-related deaths among American men. The development of metastatic castration resistant PCa (mCRPC) is the current clinical challenge. Antiandrogens such as Enzalutamide (ENZ) are commonly used for CRPC treatment. However, patients with androgen receptor (AR)-negative tumors do not respond to ENZ, while AR-positive tumors frequently develop resistance, limiting the long-term efficacy of this therapy. This study investigates the efficacy of neutral sphingomyelinase 2 (n-SMase2) inhibition by DPTIP, both alone and in combination with ENZ, as a therapeutic strategy for mCRPC. In vitro assays were conducted to determine the half-maximal inhibitory concentration (IC₅₀) of DPTIP and ENZ in mCRPC cells. The effect of these treatments on cell proliferation, migration, and colony formation was assessed. The antitumor effect of DPTIP was also evaluated in a preclinical PCa mouse model. Elevated n-SMase2 expression was observed in PCa patients compared to normal subjects at both mRNA and protein levels. In CWR-R1ca and PC-3 cells, DPTIP had IC₅₀ values of 10.31 and 14.57 µM, while ENZ had IC₅₀ values of 33.7 and 81 µM, respectively. Combined treatment significantly suppressed cell proliferation, colony formation, and migration of mCRPC cells. Mechanistically, the ERK1/2 activity and the expression of nSMase2 and NF-kB p65 were inhibited by DPTIP. The in vivo combination of DPTIP and ENZ reduced tumor size and weight more effectively than either drug alone, without significant changes in body weight. This study highlights the therapeutic potential of targeting n-SMase2 for mCRPC. Inhibition of n-SMase2 using DPTIP, both as a standalone treatment and in combination with ENZ, effectively suppressed the growth and migration of mCRPC cells. These findings suggest a promising novel approach to treating mCRPC and warrant further investigation in clinical settings.

Introduction

Prostate cancer (PCa) is one of the leading causes of cancer-associated deaths in American men [1]. Androgen signaling, mediated via androgen receptor (AR), is the main driving force for PCa progression and metastasis [2]. Systemic therapies have been used as an option for PCa management. However, chemotherapy is the ultimate solution, especially in patients with castration resistant PCa (CRPC) [3]. Although the circulating androgen level is extremely low in these patients, it is still sufficient for inducing AR signaling and maintaining tumor cell growth. The first-line therapeutic option for treating CRPC patients is anti-androgen therapy, including Enzalutamide (ENZ, MDV3100; Xtandi^®). However, clinical responses to ENZ vary significantly. Up to 25% of patients develop de novo resistance against ENZ, and other patients who initially responded are more likely to develop acquired resistance within months of treatment [4]. The development of metastatic CRPC (mCRPC) warrants the discovery of new therapies for treating mCRPC patients.

Sphingomyelinases catalyze the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Neutral sphingomyelinase 2 (n-SMase2), a subfamily of sphingomyelinase, is associated with cellular stress response, ceramide-mediated apoptosis, inflammation, neurological disorders, sEV biogenesis, cancer development, and metastasis [5-8]. Recent n-SMase2 inhibitor, 2,6-dimethoxy-4-(5-phenyl-4-thiophen-2-yl-1H-imidazole-2-yl)-phenol (DPTIP) inhibits the biogenesis of small extracellular vesicles in different pathophysiological conditions [9].

The drug combination is a highly effective approach to tackle the heterogeneity of prostate cancer and reduce drug resistance development. For example, ENZ was combined with different agents to target aggressive cancer cells [10,11]. Using a drug repurposing strategy, a combination of vasodilator hydralazine with ENZ showed a synergistic effect on PCa cells [11]. In another study, combining HIF-α inhibitor NLG207 with ENZ was more effective in pre-clinical PCa mouse models of ENZ resistance [12]. A novel nonpharmaceutical of camptothecin (CRLX101) combined with ENZ was also used in a clinical trial to treat CRPC patients (NCT03531827). In 2023, the FDA approved the combination of ENZ and PARP inhibitor talazoparib for mCRPC patients with homologous recombination repair (HRR) gene mutations (NCT03395197) [13]. Hence, novel therapies are urgently needed to target dysregulated molecular pathways in aggressive PCa cells and to reduce drug resistance.

In this study, we explored the potential of DPTIP as monotherapy and in combination with ENZ to target n-SMase2-mediated pathways in mCRPC. We investigated the expression of n-SMase2 in human tissues of PCa and normal adjacent tissues on mRNA and protein levels. The results show that n-SMase2 was overexpressed in PCa compared to normal tissues. In vitro studies revealed that the combination of DPTIP and ENZ was effective at 0.1 IC₅₀ and reached the maximum at 0.5 IC₅₀ value. When the two drugs were combined, significant n-SMase2, NF-kB, and ERk1/2 inhibitions were observed. RNA-seq analysis showed dysregulated RNA and pathways affected by DPTIP treatment compared to vehicle control cells. In a preclinical PCa model, treating mice with DPTIP reduced the tumor size and weight but did not affect the total body weight.

Materials and methods

Reagents

Cell culture media, antibiotics, and fetal bovine serum (FBS) were obtained from authenticated commercial sources. Enzalutamide (Cat#HY-70002) and 2,6-Dimethoxy-4-(5-Phenyl-4-Thiophen-2-yl-1H-Imidazol-2-yl)-Phenol (DPTIP, Cat#HY-131002) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (Cat#M6494) was purchased from ThermoFisher Scientific (Waltham, MA, USA).

Evaluating SMPD3 gene expression in PCa tissues

The expression of SMPD3 transcript in human PCa tissues was evaluated by retrieving data available from the online UALCAN based on the TCGA database (https://ualcan.path.uab.edu/) according to Chandrasekhar et al. [14,15]. The transcript per million (TPM) enrichment analysis was used for reporting gene expression according to the sample type, Gleason score (GS), nodal metastasis status and molecular signature.

Cell culture

PCa cell lines PC-3M, PC-3, and CWR-22RV1, in addition to RWPE1 normal cells, were obtained from the American Type Culture Collection (ATCC). CWR-R1ca was purchased from Millipore Sigma (Burlington, MA, USA). RWPE1 cells were grown in a keratinocyte serum-free medium containing 50 µg/mL bovine pituitary extract and 5 ng/mL human epidermal growth factor. The normal prostate epithelial PNT1A cells were maintained in RPMI-1640 medium with 2 mM L-glutamine as described [16]. PCa cells were grown in DMEM medium supplemented with 10% FBS and antibiotics and maintained in a humidified incubator at 37°C with a 5% CO₂ supply.

Cell proliferation assay

Cytotoxicity assay was performed to measure the effect of the drugs on cell proliferation in the control and treated cells. The cells were seeded in a 96-well plate and treated with different concentrations of DPTIP and ENZ for three days. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (ThermoFisher Scientific) was added into each well, after which the formazan crystals were solubilized, and the optical density was measured, using a spectrophotometric plate reader (AccuSkan FC plate reader, ThermoFisher Scientific, Waltham, MA) at 450 nm. The half-maximal inhibitory concentration (IC₅₀) values for each drug were calculated using GraphPad Software. The difference between the control and treated cells was expressed as a percentage of change.

Colony-formation assay

To assess the effect of ENZ and DPTIP on colony formation, CWR-R1Ca, CWR-22RV1, PC-3, and PC-3M were seeded in 6-well plates (500 cells/well). After 2 days, they were treated with ENZ and DPTIP at various concentrations and kept in a cell incubator for another two weeks. The cells were fixed with 4% paraformaldehyde for 10 minutes and then stained with 0.5% crystal violet for 20 minutes. The stained colonies were rinsed with tap water and left overnight to dry out. The number of colonies was then calculated using ImageJ software (US National Institutes of Health).

Cell migration assay

The Boyden chamber assay was performed to assess the effect of DPTIP and ENZ on cell migration of mCRPC cells according to our prior study [17]. Briefly, about 1.0 × 10⁵ cells were suspended in serum-free medium provided with filtered 0.1% BSA and seeded in the upper transwell chamber for 24 h with 0.5 IC₅₀ of DPTIP or ENZ, while the lower one contained complete 10% FBS medium. The migratory cells on the bottom of each insert were fixed in 4% paraformaldehyde solution, stained with 0.5% crystal violet, washed twice with water, and kept at room temperature to dry. The migrated cells were counted and photographed. The wound-healing assay was performed on mCRPC cells in another set of experiments. The cells were grown in 6-well plates until they reached 80-90% confluency, after which a pipette tip was used to create a scratch wound on the cell monolayer. Images of the wound were captured at various time points (0-48 hours) and the relative width of the wound was measured by ImageJ Software. The changes in wound width (µm) were calculated by dividing the average wound width at each time point by the initial wound width.

Immunoblot analysis

After the treatment of cells with DPTIP and ENZ, alone or in combination, for 24 and 48 hours, the cells were rinsed with PBS and lysed. Protein expression was analyzed by separating 30 µg protein lysates using SDS-PAGE and transferring the resolved proteins onto PVDF membranes. Non-specific binding was blocked with 5% BSA, and the blots were then incubated overnight with anti-n-SMase2 primary antibodies (Cat#orb158038) purchased from Biorbyt (Durham, NC, USA). Anti-NF-kB (Cat#3034), anti-ERK1/2 (Cat#4696), anti-phosphorylated ERK1/2 Thr202/Tyr204 (Cat#4376), anti-AR (Cat#5153), and anti-AR-V7 (Cat#19672) antibodies were purchased from Cell signaling Technology (Danvers, MA, USA). The blots were then washed and incubated with the appropriate secondary antibodies for 1 hour at room temperature. The protein signal was developed using a Clarity ECL Western Blotting substrate kit and visualized by Bio-Rad ChemiDoc imager (Bio-Rad, Hercules, CA, USA).

Immunohistochemistry (IHC)

The human tissue microarray (TMA) slide comprising 100 cases of PCa tissue cores, 7 BPH, and 11 standard and tumor-adjacent tissue cores were purchased from commercially available sources (US Biomax, Inc., Derwood, MD). The clinical characteristics of PCa patients are provided in Table S1. The IHC technique was performed according to our previous study [18]. In brief, tissue sections were de-waxed in two cycles of xylene and rehydrated in a descending series of ethanol solutions. The TMA slide was then heated in ethylenediaminetetraacetic acid (EDTA) buffer (pH 8.0) for 25 minutes. The slide was incubated in 3% peroxide for blocking tissue endogenous peroxidase activity. After thoroughly washing and serum blocking, the slide was incubated overnight at 4°C with n-SMase2 primary antibody (Santa Cruz Biotechnology). The developed antigen-antibody complex was detected by a commercially available Vectastain Elite ABC HRP kit (Vector Laboratories, Burlingame, CA, USA). The slide was then counterstained with hematoxylin and mounted in a permanent mounting medium. The developed protein signals were acquired using an AmScope microscope with a camera (AmScope, Irvine, CA). The stained tissues were blindly examined, and the histochemical score (HSC) was calculated as reported [19].

RNA-Seq analysis

To identify unique coding and non-coding RNAs associated with n-SMase suppression, total RNA was extracted from PC-3M cells treated with 0.1 IC₅₀ DPTIP in addition to vehicle control cells for 24 hours. For each sample, 1 µL of the final RNA eluate was used to measure mRNA concentration by Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). RNA libraries were constructed with the CleanTag Small RNA Library Preparation Kit (TriLink, San Diego, CA) according to the manufacturer’s protocol. The final purified library was quantified with High Sensitivity DNA Reagents and High Sensitivity DNA Chips (Agilent Technologies). The libraries were pooled, and the 140 to 300 bp region was size selected on 8% TBE gel (Invitrogen). The TailorMix HT1 qPCR assay (SeqMatic, Fremont, CA) was used, followed by a NextSeq High Output single-end sequencing run at SR75 using NextSeq 500/550 High Output v2 kit according to the standard protocol (Illumina, San Diego, CA). Sequencing data was uploaded into the UCSC analysis pipeline and analyzed [20]. RNA-seq analysis was performed at the Tulane University Aging Center, New Orleans, LA, USA. For a given comparison, a positive fold change value indicates an increase in expression, while a negative fold change indicates a decrease in expression. Pathway analysis was performed using Gene Set Enrichment Analysis (GSEA) and Gene Ontology (GO).

RNA isolation and gene expression

Total RNA from PCa cells was extracted using TRIzol Reagent (Invitrogen, Cat. #15596026) according to the manufacturer’s protocol. RNA was quantified on a NanoDrop spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized using oligo(dT) and random primers approach using iScript Reverse Transcription Supermix (Cat. #170884, Bio-Rad, Hercules, CA) PrimerQuest tool from Integrated DNA Technologies was utilized to design primers for the gene expression, with the GAPDH gene serving as an internal reference. The sequences of primers used int his study are presented in Table S2. Quantitative Real-Time polymerase reaction was performed using 2× SYBR Green qPCR Master Mix (Cat. #B21202, Selleck, Houston, TX) under the following conditions: denaturation: 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 30 s using qPCR (QuantStudio 3, Applied Biosystems, Waltham, MA). Relative gene expression was determined using the ΔΔCT method. The change in gene expression was calculated as a relative fold difference regarding housekeeping gapdh gene.

Establishment of cell-expressing luciferase

Stable luciferase-bearing CWR-R1Ca cells were established as described [21]. Lentiviral particles carrying luciferase from Kerafast (Boston, MA, USA) were briefly transduced into PCa cells, followed by puromycin selection. Cells with luciferase bioluminescence were examined using the Perkin Elmer InVivo machine (IVIS; PerkinElmer, Waltham, MA).

Preclinical study

Animal experiments were carried out using an IACUC protocol approved by the Institutional Animal Care and Use Committee of the University of Louisiana at Monroe (IACUC approval #21AUG-ZE-01). About 4-6-week-old male nude mice were purchased from Envigo (Indianapolis, IN, USA). About 50 µL of CWR-R1ca-luciferase cells (2 × 10⁶ cells) mixed with an equivalent volume of Matrigel^TM (BD Bioscience) kept on ice were injected into the dorsal flank of each mouse. The individual and combined drugs were intraperitoneally administered daily for 3 weeks. Animals were divided into 4 groups: 5 mice each. Group 1 received a vehicle; Group 2 received 10 mg/Kg ENZ, as reported [22,23]; Group 3 received 10 mg/Kg DPTIP according to a previous study [9]; and Group 4 received 10 mg/Kg ENZ combined with 10 mg/Kg DPTIP. After 1 and 3 weeks from initiating the treatment, D-luciferin (10 µL/g body weight) was intraperitoneally injected, and after 10 minutes, bioluminescent images were acquired using an In Vivo Imaging System (IVIS; PerkinElmer, Waltham, MA). During the experiment, mice were weighed, and tumor volume was calculated twice a week using the formula π/6 (L × W²) as described [24]. The tumor mass was collected, and one piece was fixed in 10% neutral formalin for histopathological studies, while the other pieces were snap-frozen for biochemical analyses.

Statistical analysis

Data presented as mean ± standard error of the mean (SEM). Statistical comparisons between experimental and control groups were performed using an unpaired Student’s t-test. For multiple group comparisons, one-way analysis of variance (ANOVA) was used, followed by Tukey’s post-hoc test to correct for multiple comparisons. Statistical significance was determined at P<0.05. All statistical analyses were conducted using GraphPad Prism version 10.10.0 for Windows (GraphPad Software, Boston, Massachusetts, USA).

Results

Expression of n-SMase2 in human PCa tissues

We investigated the expression n-SMase2, encoded by SMPD3 gene, in available human cancers (Figure S1) and in PCa tissues and normal adjacent cores on both the mRNA and protein levels using TCGA available data and human tissue microarray (TMA). SMPD3 transcript was upregulated in pancreatic cancer, PCa, pheochromocytoma and sarcoma compared to normal tissues. TCGA data analysis showed that SMPD3 was upregulated in PCa (P<0.0001) compared to normal tissues (Figure 1A). Notably, SMPD3 expression was elevated across different tumor stages using Gleason score (GS) 6 to 9 (P=0.019) when compared to normal individuals. However, we found no statistically significant differences in lymph node involvement based on SMPD3 expression levels in the analyzed tissues. Regarding molecular signatures, SMPD3 was found to be upregulated in tissues associated with ETS-related gene (ERG) fusions, PEA3 subfamily ETV1 & 4, and Friend Leukemia Integration 1 (FLI1) gene fusions. On the protein level, the immunostaining of TMA tissue cores with an n-SMase2 antibody revealed high expression in tissues collected from patients with GS 7, moderate expression in GS 9 tissues, and low expression in normal and benign hyperplastic prostate tissues (Figure 1B). Immunohistochemical score (IHS) was used to evaluate the protein expression in PCa tissues as reported [19,25]. The IHS of n-SMase2 was high in PCa, confirming the mRNA level results. Despite the findings indicating varying levels of n-SMase2 expression, there was no significant difference in protein expression based on age at diagnosis, Gleason score, lymph node involvement, or metastatic status (Figure 1C). This suggests that n-SMase2 may play a role in the pathogenesis of PCa at early stages, independent of these clinical factors. These observations support the notion of targeting n-SMase2 as an evidence-based therapeutic approach, potentially providing a strategy for intervention in the early progression of the disease.

Figure 1.

Expression of n-SMase2 in human PCa tissues. A: Expression of n-SMase2 gene SMPD3 transcript retrieved from TCGA data of PCa compared to normal tissues. B: The protein expression of nSMase2 was evaluated by immunohistochemistry in tissue microarray slide comprising 100 cases of PCa tissue cores, 7 BPH, and 11 normal and tumor adjacent tissues cores. The protein expression was represented according to Gleason score (GS). Another slide was incubated only with secondary antibody and kept as a negative control (CTR). C: The immunostaining of nSMase2 was expressed as immunohistochemical score (IHS) which was used to compare PCa with normal tissues, age at diagnosis, Gleason score (GS), pathological stages (T2-4), lymph node metastatic status and distant metastasis (Met). Quantification of immunohistochemical score of n-SMase2 staining. Magnification is 50 µm (solid line) and 20 µm (dashed line). Data considered significant at P<0.01 regarding control groups. N: number; N0/N1: involvement of lymph nodes.

Effect of DPTIP combination with ENZ on mCRPC cells

To establish effective dosing parameters for DPTIP and ENZ, we first determined the IC₅₀ values for the two drugs across multiple mCRPC cell lines, including PC-3, PC-3M, CWR-22RV1, and CWR-R1Ca, as well as normal prostate epithelial (PNT1A) cells. Cells were treated with different concentrations of DPTIP or ENZ at a concentration range from 0-250 µM for up to 72 hours, and IC₅₀ values were calculated based on cell viability assay [18] as shown in Table 1. The IC₅₀ values for PC-3 and its highly metastatic derived PC-3M cells recorded the highest values compared to CWR-22RV1 and CWR-R1ca cells (IC₅₀ value range from 31 to 34 µM) because they lack AR as a constitutive target. The IC₅₀ value of ENZ was the highest in normal prostate PNT1A cells, which indicates the drug’s selectivity towards tumor cells. The same pattern was observed in PNT1A treated with DPTIP (IC₅₀=27.1 µM) compared to PCa cells. Based on these results, concentrations corresponding to 0.1 and 0.5 IC₅₀ were selected for subsequent experiments. To assess the potential synergistic effects of DPTIP and ENZ, mCRPC cells were treated with drug individually at 0.1 and 0.5 IC₅₀ as well as in combination. As shown in Figure 2, the most practical combination of the two drugs was observed in CWR-22RV1 (Figure 2A) and CWR-R1Ca (Figure 2B) cells at the concentration of 0.5 IC₅₀ of either drug (P<0.001) when compared to individual treatments. To understand the differential response to DPTIP treatment, Western blotting analysis examined the expression of n-SMase2 in different PCa and normal cells. The protein expression was markedly low in RWPE1 normal cells, moderate in CWR-22RV1 and CWR-R1Ca cells, and high in PC-3 and PC-3M cells (Figure 2C, 2D). The expression of AR and its variant AR-V7 was also validated in these cells by immunoblot analysis.

Table 1.

Determination of IC₅₀ for DPTIP and enzalutamide in mCRPC cells

Cell line/IC50	DPTIP (µM)	Enzalutamide (µM)
CWR-22RV1	9.80	31.8
CWR-R1ca	10.31	33.7
PC-3	14.57	81
PC-3M	15.27	95
PNT1A	27.14	220

Figure 2.

Cytotoxic effect of treatment of mCRPC cells with combined drugs. (A, B) CWR-22RV1 (A) and CWR-R1Ca (B) cells were treated with 0.1 and 0.5 of IC₅₀ of DPTIP (D) or enzalutamide (E), individually and in combination, for 72 hours. Cell viability assay was performed as indicated and the percentage of O.D. change relative to control cells was calculated. (C) Western blot analysis was performed for AR, AR-V7 and n-SMase2 expressions. β-actin was used as loading control. (D) The endogenous expression of n-SMase2 was calculated relative to the house keeping protein and expressed as arbitrary unit compared to the control cells. *, # denote significance at P<0.05 relative to vehicle control cells and individual treatment, respectively.

Effect of DPTIP and ENZ on colony formation and cell migration

To assess the long-term anti-proliferative effects of DPTIP and ENZ, colony formation assay was performed. AR-expressing CWR-22RV1 cells were treated with 0.1 and 0.5 IC₅₀ of DPTIP and ENZ, either alone or in combination, and allow to grow for up to 14 days. As shown in Figure 3, the treatment of cells with individual drugs decreased the number and size of colonies, and this effect increased at 0.5 IC₅₀ value of each drug. Interestingly, the drug combination at 0.5 IC₅₀ value showed a 90% decrease in the number and size of colonies compared to individual drug treatments. The same effect was observed in AR-naïve PC-3 cells, where the drug combination at 0.5 IC₅₀ value showed a 96% colony number reduction. Regarding cell migration, a Boyden chamber assay was performed on CWR-R1Ca and PC-3 cells (Figure 4). The individual treatment of cells with 0.5 IC₅₀ values of ENZ or DPTIP decreased migratory cells by 50 and 68%, respectively. Cells treated with the combined drugs showed an 85% reduction in cell migration compared to single treatment. Another confirmatory experiment was conducted on PC-3 cells using a scratch assay. Cells were seeded in a 6-well plate, and once they reached confluency, a scratch assay was performed. Cells treated with individual or combined drugs and photographed after 0, 24, and 48 h of treatment. Treating cells for 48 h with 0.5 IC₅₀ of the combined drugs reduced cell motility by 35% and 72% compared to single treatment and vehicle control cells, respectively (Figure S2).

Figure 3.

Effect of treatment of mCRPC cells with DPTIP and Enzalutamide on colony formation. CWR-22RV1 and PC-3 cells were treated with 0.1 and 0.5 IC₅₀ of DPTIP (D) and Enzalutamide (E), and their combinations for 14 days, fixed, stained and dried out for imaging. The number and size of cell colonies were counted and presented as mean + SEM as indicated in bar graphs. *, # depict statistical significance at P<0.05 regarding vehicle control or individual treatment, respectively. Each treatment was conducted in duplicate, and the experiment was independently repeated twice.

Figure 4.

Effect of DPTIP and ENZ on mCRPC cell migration. A: About 5 × 10⁴ cells were seeded in upper insert in FBS-depleted medium while complete medium was added to lower wells. Cells were treated with 0.5 IC₅₀ of DPTIP and ENZ, and their combinations for 36 h. The migrated cells were fixed, stained, and imaged. B: The migrated cells were counted and presented as percentage of migratory cells. *, #, ≠ depict significance at P<0.05 compared to vehicle control, ENZ and DPTIP, respectively. NS: Nonsignificant. Magnification was 100×.

Effect of DPTIP on ERK1/2 activity and NF-kB expression

To confirm the inhibitory effect of DPTIP on the protein expression of n-SMase2, immunoblotting analysis was performed on PC-3M cells which have a relative high endogenous n-SMase2 level (Figure 5) compared to low n-SMase2 level in CWR-R1Ca cells (Figure S3). Treatement of PC-3M cells with DPTIP for 24 and 48 h exhibited a significant inhibition in n-SMase2 expression when treated with DPTIP compared to ENZ-treated cells. This effect was augmented after the two drugs were combined at 0.1 or 0.5 IC₅₀ (Figure 5). Since MEK-ERK and NF-kB singlaing pathway contribute to deveopment and progression of mCRPC and reported as therapetuc targets [26], we selected to assess the inhbitory effect of DPTIP on these pathways. While the activity of ERK1/2 showed a relative inhbition in cells treated with either drug individually, the ERK1/2 activity was markedly decreased when the two drugs were combined. This effect was increased after 48 h of drug tretament. NF-kB expression was reduced after DPTIP but not ENZ treatment and the drug combination showed a further reduction at 0.5 IC₅₀ of both drugs. Treatment of CWR-R1Ca cells for 24 h had different pattern of protein expression comapred to PC-3M as shown in Figure S3. The expression of n-SMase2 was decreased 0.5 IC₅₀ of the combined drugs while NF-kB expression was reduced when cells treated with 0.5 IC₅₀ of DPTIP and reached the maxium with 0.5 IC₅₀ of ENZ treatment.

Figure 5.

Molecular effect of DPTIP and Enzalutamide treatments on PC-3M cells. A: Cells were treated with 0.1 and 0.5 IC₅₀ of DPTIP (D) and Enzalutamide (E), and their combinations for 24 and 48 h. Cell lysates were collected and subjected to Western blot analysis. The membrane-bound proteins were incubated with antibodies against nSMase2, phosphorylated and total ERK1/2, and NF-kB p65. B: The relative expression was calculated as fold change relative to control cells and represented as mean ± SEM as indicated in bar graphs. *, # depict statistical significance at P<0.05 regarding vehicle control or individual treatments, respectively.

The treatment of mCRPC cells with DPTIP induces gene dysregulation and targets multiple signaling pathways

The treatment of PC-3M cells with 0.1 IC₅₀ DPTIP for 24 h changes the expression of coding and non-coding RNAs. The most upregulated and downregulated genes are listed in Table S3 and illustrated in Figure 6. Among the most upregulated genes as shown in Table 2, exonuclease 5 (Exo5) which showed a 14-fold increase in DPTIP-treated cells. The function of Exo5 is to assist in repairing nuclear DNA damage caused by external stimuli [27]. Additionally, fibulin 7 (FBL7) and killer cell lectin-like receptor C1 (KLRC1) were both upregulated by 10-fold. Conversely, coding mRNA Nox organizer 1 (NOXO1) and non-coding long intergenic non-protein coding RNA 482 (LINC00482) were significantly downregulated. Previous studies have shown that the Nox1 inhibitor NOS31 selectively inhibits Nox1-mediated reactive oxygen species (ROS) generation, which is closely associated to NOXO1 function in colon cancer cells [28]. Moreover, a group of researchers identified a set of upregulated non-coding RNAs, including LINC00482, which was associated with biochemical relapses in PCa patients [29]. Figure 6A and 6B shows the identified molecular pathways and their associated genes in cells treated with DPTIP. These pathways include E2F, G2M, MYC, and estrogen response pathways. To further validate these findings, we evaluated the expression of the most relevant genes associated with PC-3M-treated cells with DPTIP, ENZ and their combinations (Figure 6C). qPCR analysis revealed that FBLN7, KLRC, C10orf90 and TBX4 genes were upregulated while NOXO1 was downregulated after DPTIP treatment for 48 h compared to vehicle control cells.

Figure 6.

RNA-seq and molecular pathways in mCRPC cells treated with DPTIP. A: Visualizing GSEA enrichment results for DPTIP-treated vs. vehicle control PC-3M cells using ggplot2. B: Top five non-redundant enriched terms for the GO class molecular function. C: Quantitative Real-Time PCR analysis was performed as indicated and presented as fold change to cells received vehicle. *, # depict significance at P<0.01 regarding vehicle control cells.

Table 2.

Top listed up- and down-regulated RNAs after treatment of PC-3M cells with DPTIP

	Gene	Description	Log FC	RNA
1	Exo5	Exonuclease 5	+14	Coding
2	FBLN7	Fibulin-7	+10	Coding
3	KLRC1	Killer Cell Lectin Like Receptor C1 (NKG2A)	+10	Coding
4	C10orf90	The fragile-site associated tumor suppressor	+9	Coding
5	TBX4	T-Box Transcription Factor 4	+8	Coding
6	HTR2C	5-Hydroxytryptamine Receptor 2C	+5	Coding
7	NOXO1	Nox organizer 1	-5	Coding
8	LINC00482	Long Intergenic Non-Protein Coding RNA 482	-7	Non-coding
9	GIMAP2	GTPase, IMAP Family Member 2	-8	Coding
10	DPT	Dermatopontin	-8	Coding

RNA-seq analysis of PC-3M cells treated with DPTIP compared with untreated cells.

Effect of DPTIP and ENZ on tumor size in nude mice

To validate the in vitro findings, we conducted in vivo experiments using a nude mouse xenograft model. Male nude mice were injected subcutaneously with CWR-R1Ca cells. Once the tumor size became palpable, the mice were treated with intraperitoneal injections of DPTIP and ENZ at a dose of 10 mg/kg/mouse, either individually or in combination, for 3 weeks (Figure 7A). Prior to sacrifice, D-luciferin was intraperitoneally injected, and live images were acquired (Figure 7B). Mice treated with single DPTIP or ENZ, revealed significant tumor size reductions of 29.6 and 34.3%, respectively, compared to vehicle-treated control (Figure 7C). The combination of DPTIP and ENZ further reduced tumor size by 45.3%. Notably, the two drugs did not elicit any significant changes in body weight, which implies the safety levels of the injected doses of single and combined drugs (Figure 7D). After the mice were sacrificed, tumors were resected and weighed. DPTIP and ENZ showed a significant reduction in tumor weight, and although their combination further decreased the tumor weight, it did not reach statistical significance when compared to either drug alone (Figure 7E).

Figure 7.

Antitumor effect of DPTIP in preclinical PCa model. (A) A representative experimental design for mice treatment. Mice were injected with CWR-R1ca-luc cells (2 × 10⁶ cells). After 2 weeks, intraperitoneal injection of Enzalutamide (Enz) and DPTIP (10 mg/Kg each) was initiated and continued for another 3 weeks. (B) In Vivo Imaging was acquired at the baseline (week 1) and after 3 weeks of drug treatment. (C, D) Tumor size (C) and total body weight (D) were recorded twice a week for 3 weeks. (E) Tumor mass was excised after mice sacrifice and weighed. * depicts significance at P<0.05. NS: non-significant data.

Discussion

Our findings highlight the significant role of n-SMase2 and its encoded gene SMPD3 in PCa, particularly its overexpression in PCa, especially at GS 7, in comparison to other tissues. The combination of the nSMase-2 inhibitor DPTIP with enzalutamide (ENZ) shows promise, with optimal efficacy observed at 0.5 IC₅₀ for both drugs. The data demonstrate that treatment with either DPTIP or ENZ effectively inhibits cell proliferation and migration in mCRPC cells, with even greater effects observed with the combination treatment. Furthermore, the in vivo studies with CWR-R1Ca xenografts support the notion that DPTIP contributes to tumor size reduction, indicating its potential as a therapeutic agent in PCa treatment.

High ceramide levels are associated with aggressive PCa forms, failure in lowering testosterone levels in metastatic hormone-sensitive PCa, and shorter survival time in mCRPC [30]. Sphingomyelinases catalyze the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. n-SMase2, a subfamily of sphingomyelinase, is associated with cellular stress response, ceramide-mediated apoptosis, inflammation, neurological disorders, small extracellular vesicle (sEV) biogenesis, and cancer development and metastasis [5-8]. During sEV biogenesis, a double membrane invagination of cell plasma membrane forms multivesicular bodies (MVB), which are either endosomal sorting complexes required for transport (ESCRT)-dependent or -independent process [31]. Therefore, n-SMase2 is a potential therapeutic target in different diseases where the biogenesis of sEVs is optional for using the ESCRT-independent pathway [32]. Inhibition of n-SMase2 presents a novel and selective anti-tumor strategy in PCa. This approach is particularly compelling given its broader implications for other diseases. For instance, in Parkinson’s disease, the inhibition of n-SMase2 by cannabinol has been shown to reduce extracellular vesicle (EV) release and alpha-synuclein accumulation, leading to improved motor function in mice [33]. In another study, the inhibition of n-SMase2 by phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl) carbamate in neurons and oligodendrocytes decreased EVs secretion and activated microglia cells after acute brain injury [34]. DPTIP was reported to specifically inhibit n-SMase2 and suppress EV biogenesis, which reduces cytokine production in the liver and immune cell infiltration in the brain [9]. A research group developed an orally bioavailable and brain-penetrable prodrug called P18, which exerted 4-fold higher plasma and brain exposures versus the parent compound DPTIP [35]. These findings suggest the role of n-SMase2 in the pathogenesis of different diseases and justify the molecular targeting of n-SMase2 as an evidence-based approach for offering a new strategy for treating mCRPC patients.

Most FDA-approved drugs primarily target patients with localized CRPC and metastatic castration-sensitive PCa (mCSPC) [36]. The landscape for treating these conditions is continually evolving, with ongoing research aimed at improving outcomes for patients at various stages of the disease. Repurposing or developing novel drugs specifically targeting mCRPC remains an unmet clinical need. While ENZ exhibits variable efficacy depending on androgen receptor (AR) status in mCRPC cells, our findings demonstrate that DPTIP has a significant cytotoxic effect regardless of AR status. This suggests that DPTIP could be an effective treatment option across different subtypes of PCa, potentially overcoming some of the limitations associated with current therapies that are AR-dependent.

The consistent efficacy of DPTIP, as indicated by the recorded IC₅₀ values across various PCa cells, underscores its potential as a promising candidate for further development in the treatment of mCRPC. This could pave the way for more effective therapeutic strategies that are less reliant on AR pathways, addressing a critical gap in the current treatment landscape. Interestingly, DPTIP showed a relative selectivity against PCa cells compared to normal PNT1A prostate epithelial cells. Therefore, the combination of DPTIP and ENZ could bring more benefits to mCRPC patients. The combination of ENZ with other therapeutic agents has been reported in different studies. For example, the combination of cholesterol-lowering simvastatin with the FDA-approved darolutamide inhibited CRPC cell growth via suppression of cell cycle regulators [37]. The treatment of CRPC cells with chemokine receptor CXCR7 inhibitor and ENZ suppressed cell growth and proangiogenic signaling pathways [38]. The combination of ENZ and sorafenib decreased cell proliferation-induced apoptosis in vitro and reduced tumor size in castration-resistant LNCaP-xenograft model [39]. It is well known that the aldo-keto reductase 1C3 (AKR1C3) enzyme is associated with intratumor androgen biosynthesis and development of CRPC status, and therefore, its pharmacological inhibition decreased cell growth and sensitized CRPC cells to ENZ [40]. Another research group used Akt pharmacological inhibitor AZD5363 in ENZ-resistant cells, and it reduced tumor size in the CRPC mouse model [41].

The combination treatment of mCRPC cells with both drugs at 0.5 IC₅₀ significantly decreased the number of colonies and migratory cells compared to treatment with either drug alone. A group of researchers found that AR-positive PCa cells displayed sigmoidal dose-response curves for ENZ with IC₅₀ values ranging from 19 to 45 µM as compared to AR-naïve cells, which had higher IC₅₀ values [42]. This study is comparable with the IC₅₀ values of ENZ-treated mCRPC cells recorded in our study. In an independent study, the inhibition of n-SMase2 by DPTIP downregulated mRNAs for a set of genes involved in transendothelial migration [43]. The same study showed that regulatory T cells inhibit n-SMase2 activity to prevent T-cell migration into intestinal tumors by decreasing chemokine secretion and vascular cell adhesion molecule 1 (VCAM1) expression. Exploring these gene targets may provide insights into the pathways affected by n-SMase2 inhibition and enhance our understanding of mCRPC behavior. Chemotherapeutic agents such as etoposide and paclitaxel activate n-SMase2 and induce cell apoptosis in PCa cells via protein kinase C delta (PKC-delta)-dependent pathway to mediate their cytotoxic effects [44]. In another study, Clarke et al. reported n-SMase2/S6K-axis as a significant component of all-trans-retinoic acid (ATRA)-induced cell cycle arrest in breast cancer cells [45]. When multiple myeloma and hepatocellular carcinoma cells were treated with n-SMase2 inhibitor cannabinol, the drug decreased cell proliferation and induced cell apoptosis [46,47]. When tissue-type plasminogen activator (tPA) activates smooth muscle cells and ECV-304 carcinoma cells, tPA promotes phosphorylation of ERK1/2 proteins through the sphingomyelin/ceramide pathway, which induces cell proliferation [48]. It was also reported that the blockade of the NF-kB pathway in cancer cells is associated with the inhibition of cell proliferation, migration, invasion, angiogenesis, drug resistance, and metastasis [49,50]. Thus, the inhibition of these pathways by DPTIP aligns with the significant inhibition of cell growth, colony formation, and migration observed in mCRPC cells.

Our results demonstrate that the treatment of mCRPC cells leads to gene dysregulations associated with multiple signaling pathways. One notable example of a dysregulated gene is EXO5. The knockout of EXO5 in PCa cells impaired homology-directed recombination repair (HDR) and caused androgen-induced genomic instability [51]. The upregulation of EXO5 (14-fold) in our results suggests that DPTIP restored the tumor suppression function of this gene. It was reported that another dysregulated gene, fibulin-7 (Fbln7), inhibits tube formation [52]. These studies implicated the anti-angiogenic effect of Fbln7 in cancer, which aligned with our RNA-seq results (10-fold up). NKGA2 (KLRC1) is associated with improved survival and responsiveness to programmed death ligand 1 (PD-L1) blockade in bladder cancer with high CD8⁺ T-cells [53]. The upregulation of fragile-site associated tumor suppressor (FATS or C10orf90) mRNA could be a prognostic marker for improved clinical outcomes in patients with non-small cell lung carcinoma (NSCLC) who received cisplatin-based chemotherapy [54,55]. Another gene called Nox organizer 1 (NOXO1) belongs to the NADPH oxidase family and is associated with generating reactive oxygen species [56]. Long non-coding RNA LINC00482 inhibits tumor-associated inflammation and angiogenesis in bladder cancer and tumor metastasis in NSCLC [57,58]. Our results show that NOXO1 and LINC00482 were downregulated after DPTIP treatment, which may validate their contribution to the n-SMase2 blockade. However, further studies are warranted to validate these genes in mCRPC cells.

The preclinical results show that mice treated with either DPTIP or ENZ revealed a significant reduction in tumor size. Notably, this effect was further enhanced when the two drugs were combined. Importantly, there were no observed effects on body weight, indicating that the combination treatment is well-tolerated. The research group that developed DPTIP reported that it exhibits potent non-competitive and selective inhibition against n-SMase2. Additionally, DPTIP has demonstrated metabolic stability in both mouse and human liver microsomes, indicating its potential for effective preclinical use without rapid degradation [9]. The inhibition of ceramide-S1P signaling with sphingosine kinase inhibitors enhanced ENZ efficacy in PCa cells through sterol regulatory element-binding protein 1 (SREBP)-induced lipotoxicity [59].

Although DPTIP has an excellent antitumor profile in vitro, the in vivo studies did not fully recapitulate the results observed in cell culture models. Several factors may contribute to this discrepancy, including drug pharmacokinetics, metabolic stability, and the need for dose adjustments [60]. These factors underscore the complexity of translating in vitro results to in vivo efficacy. In a study conducted on LNCaP-xenografted mice treated with ENZ at doses of 1, 10, and 50 mg/Kg daily, significant tumor size reduction was observed. Specifically, the 10 mg/kg dose resulted in a notable decrease in tumor size after 24 days of treatment, while the 50 mg/kg dose achieved similar results after 28 days [61].

One of the limitations of this study is that targeted downstream signaling pathways associated with the drug combination require further investigation. Additionally, the relatively short treatment duration in mice and the specific phenotype of prostate cancer (PCa) cells used in the study may limit the generalizability of the findings. Given the potential role of n-SMase2 in PCa, targeting this enzyme could represent a promising therapeutic strategy. Inhibiting n-SMase2 with DPTIP might not only suppress tumor growth and reduce metastasis but also enhance overall patient outcomes. However, more extensive research is needed to fully understand the complex role of n-SMase2 in mCRPC cells and to develop more effective therapeutic interventions. Future studies should aim to explore the signaling pathways involved and evaluate different PCa models to strengthen the evidence for n-SMase2 as a viable therapeutic target.

Conclusions

Our study demonstrates that DPTIP exhibits potent anti-proliferative and anti-migratory effects on mCRPC cells in vitro and promotes tumor regression in a preclinical mouse model. Notably, the combination of DPTIP with ENZ proved to be more effective at reducing tumor size in CWR-R1Ca xenografts than either drug alone, suggesting a synergistic effect. These findings underscore the potential of DPTIP as a promising treatment candidate for mCRPC. Given the robust anti-tumor activity observed both in vitro and in vivo models, further preclinical studies and clinical investigations are warranted to explore its full therapeutic potential.

Disclosure of conflict of interest

None.