Regulatory roles of NAMPT and NAD⁺ metabolism in uterine leiomyoma progression: Implications for ECM accumulation, stemness, and microenvironment

Abstract

Uterine leiomyoma (UL), commonly referred to as benign tumors, is characterized by excessive cell proliferation, extracellular matrix (ECM) accumulation, and the presence of stem cell-like properties. Nicotinamide adenine dinucleotide (NAD⁺) metabolism, regulated in part by nicotinamide phosphoribosyltransferase (NAMPT), plays a crucial role in these pathological processes and has emerged as a potential therapeutic target. Additionally, redox signaling pathways are integral to the pathogenesis of UL, influencing the dynamics of NAD⁺ metabolism. This study sought to elucidate the regulatory functions of NAMPT and NAD⁺ metabolism, in conjunction with redox signaling, in the progression of UL, and to explore potential therapeutic strategies targeting these pathways. Evaluation of NAMPT expression in human UL tissues revealed a positive correlation between elevated NAMPT levels and increased ECM deposition, as well as the expression of stemness markers. The use of FK866 and nicotinamide (NAM), to inhibit NAMPT significantly suppressed UL cell viability and attenuated stem cell-like characteristics. Redox signaling pathways, including those associated with DNA damage, lysosomal function homeostasis, and redox-sensitive phagophore formation, were implicated in the regulation of ECM dynamics, particularly through ECM-targeted inhibition. This study highlights the pivotal roles of NAMPT, NAD⁺ metabolism, and redox signaling in the pathophysiology of UL. Targeting NAMPT, particularly through the use of inhibitors FK866 and NAM, represents a promising therapeutic approach for mitigating UL progression by modulating redox and ECM dynamics. These findings offer novel insights into UL pathogenesis and establish NAMPT as a compelling target for future clinical investigation.

1. Introduction

Uterine leiomyoma (UL), also known as uterine fibroids, is the most common pelvic tumor among reproductive women. The prevalence rate can reach 42.2 % in women over 35 years old and can be as high as 66.1 % in women aged 46 to 51 [1]. Despite its high prevalence, the pathogenesis, incidence, and risk factors of UL are not fully understood. UL often poses a significant healthcare burden on women's health and has a profound impact on healthcare costs worldwide. Symptoms include bleeding, pelvic pain, infertility, and reduced quality of life [2]. Clinical treatments have been limited to the relief of symptoms or a hysteroscopic myomectomy, but with high recurrence within 5–10 years [3]. Therefore, it is important to gain a greater understanding of the pathology and therapeutic modulation of UL as a crucial research goal.

The extracellular matrix (ECM) accumulation, specifically involving fibronectin, collagen, and proteoglycans, plays a role in modulating ECM accumulation, resulting in increased proliferation, tissue stiffness, and downstream molecular signaling pathways [4]. The ECM accumulation also creates a unique microenvironment that regulates the oxidative stress, cytokines, and chemokines, thereby promoting the progression of UL [5,6]. The high recurrence of UL was caused by the abnormal regulation of stem cell transformation in UL [7]. Simultaneously, factors involved in stem cell transformation promote paracrine effects and inducing relevant signaling pathways, resulting in the high recurrence of UL [8].

Rapidly proliferating or abnormally proliferating cells exhibit distinct energy metabolism compared to normal cells. One prominent feature is their preference for anaerobic glycolysis, commonly referred to as the Warburg effect [9]. This metabolic phenomenon involves the conversion of glucose into pyruvate, leading to the production of lactate for energy generation. The reliance on anaerobic glycolysis provides an advantageous environment for supporting the proliferation of hyperactive cells [10]. Notably, the utilization of glucose as an energy source relies on the availability of nicotinamide adenine dinucleotide (NAD) as a coenzyme for energy utilization [11].

Nicotinamide phosphoribosyltransferase (NAMPT), also known as visfatin, is a key enzyme in NAD metabolism, serving as the rate-limiting enzyme in the conversion of nicotinamide (NAM) into nicotinamide mononucleotide (NMN), which is then used to generate NAD and sustain cellular energy production [12,13]. NAMPT not only modulates NAD utilization but also exhibits cytokine and adipocytokine properties, influencing various cellular processes [14,15]. Intracellular NAMPT (iNAMPT) supports energy homeostasis by regulating NAD biosynthesis, while extracellular NAMPT (eNAMPT) can alter the microenvironment, promoting cell proliferation and inhibiting apoptosis [16,17]. However, the mechanisms regulating eNAMPT release are not yet fully understood. In rapidly proliferating cells, such as cancer cells, NAMPT expression is elevated and correlates with poor overall survival outcomes [18,19]. Moreover, NAMPT plays a role in modulating ECM accumulation and contributes to fibrosis progression in fibrotic diseases [20].

Previous studies have shown that UL tissue exhibits higher NAMPT expression compared to the myometrium [21]. However, there is a lack of comprehensive research on the regulation of NAMPT and NAD metabolism in UL, as well as its association with ECM accumulation. Our ultimate goal is to find out the novel therapeutic target that can impede UL progression by modulating NAMPT and NAD metabolism, thereby providing a novel avenue for treatment.

In this study, we demonstrate the pivotal role of NAMPT modulation in the progression of UL, highlighting its regulatory significance in disease development. We explore the effects of NAMPT-related inhibitors on key pathological features of UL, including cell proliferation, ECM accumulation, and stemness progression. By identifying NAMPT as a potential therapeutic target, our research provides valuable insights into the development of targeted treatments that may improve the management and outcomes for patients with UL.

2. Materials and methods

2.1. Reagent

The NAMPT inhibitor FK866 (13287, Cayman Chemical (Ann Arbor, MI, USA)) was dissolved in dimethyl sulfoxide (DMSO) (ECHO Chemical Co. Ltd. (Taipei, Taiwan)) at a 10 mM concentration. The NAM (N0636, Sigma-Aldrich (St Louis, MO, USA)) was dissolved in phosphate buffered saline (PBS) at a 2.5 M concentration. The NMN (NN45555, Carbosynth (Staad, Switzerland)) was dissolved in cultured medium at a 50 mM concentration.

2.2. Ethics statement

Human tissues were obtained from 30 to 45 year-old women (n = 9) who had received myomectomy treatment at the Department of Oncology, Taipei Medical University Hospital (Taipei, Taiwan). The study was approved by the Institutional Review Board and Ethics Committee of Taipei Medical University Hospital (Permit Number: N202305018).

The animal studies were conducted according to the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Taipei Medical University (No.: LAC-2019-0535).

2.3. Cell lines and culture condition

A rat leiomyoma cell line, Eker rat-derived uterine leiomyoma (ELT-3), was kindly obtained from Prof. Lin-Hung Wei (Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan). The human uterine leiomyoma (HuLM) cell line was obtained from Dr. Ayman Al-Hendy [22]. The human uterine smooth muscle (UtSMC) cell line was purchased from Promocell (C-12575, PromoCell (Heidelberg, Germany)). The ELT-3-LUC cell line was established based on a previous study [23]. The ELT-3, ELT-3-LUC and UtSMC were cultured in DMEM F12 (DFP29, CAISSON Labs (Smithfield, UT, USA)) suppled with 10 % fetal bovine serum (FBS) (35-010-CV, CORNING (Manassas, VA, USA)) and a 1 % Antibiotic-Antimycotic (30-004-CI, CORNING (Manassas, VA, USA)). The HuLM was cultured in SmGM™- 2 Smooth Muscle Cell Growth Medium −2 BulletKit™ (CC-3182, Lonza (Basel, Switzerland)).

2.4. Uterine leiomyoma and myometrium tissue collection

Uterine leiomyoma tissues (L) were collected from women aged (30–40-years-old) who were undergoing the myomectomy (n = 9) and the surrounding normal tissue were considered as myometrium tissue (M). And kept at −80 °C for further usage for Western blot to exam the NAMPT protein expression.

2.5. Uterine leiomyoma tissue array and image analysis

Uterine leiomyoma tissue array (SO806) was obtained from Biomax Inc. (Derwood, MD, USA), stained with NAMPT (1:200, 11776-1-AP, Proteintech (Rosemont, IL, USA)) and the COL1A1 (1:500, GTX112731, Genetex (Irvine, CA, USA)) antibody by RaPID Science co., ltd (Taichung, Taiwan). Images were scanned by a TissueGnostics Axio Observer Z1 microscope (TissueGnostics, Vienna, Austria) and analyzed by HistoQuest (TissueGnostics), the value of NAMPT and COL1A1 were further used for the correlation analysis.

2.6. Human tissue and gene correlation

The gene expression database of 16 African-American UL patients from the Gene Expression Omnibus database under accession number GSE31699 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE31699) [24]. By defined the myometrium group and UL group, the mRNA values were used to evaluate the correlation between NAMPT, COL1A1, MMP-9 and CD44. With the clinical characteristic from the GEO database contributor, we established the correlation between tumor weight, number of tumors and the NAMPT mRNA levels.

2.7. NAMPT activity assay

The NAMPT enzyme activity was measured by an NAMPT Activity Assay Kit (Colorimetric, ab221819, abcam (Cambridge, MA, USA)) according to the manufacturer's instructions, which required adding the NAMPT-related metabolic precursor to measure the NAMPT enzyme activity. By using the two-step activity assay protocol. Sample were added reaction mix I to convert nicotinamide to NAD⁺ after reaction at 30 °C for 30 min, used reaction mix II to measure the generated NAD⁺. Used kinetic mode to measure the wavelength of OD 450 nm every 5 min for 60 min at 30 °C. The activity was calculated by the OD values divided by the corresponding time point in the linear phase [14].

2.8. siRNA transfection

The HuLM cells (1 × 10⁵ cells/well) were seeded in a 6-well plate overnight. The transfection procedure was according to the manufacturer's instructions, 25 or 50 nM of the SMARTpool: siGENOME NAMPT sequence (M-004581-01-0005, Dharmacon (Lafayette, CO, USA)) and the siGENOME Non-Targeting siRNA pool were transfected by lipofectamine3000 (Thermo) reagent in Opi-MEM (Gibco) for 48 h.

2.9. Proliferation measurement

MTT (Thiazolyl Blue, HY-15924, Medchemexpress (Monmouth Junction, NJ, USA) powder was dissolved in PBS with a 5 mg/ml concentration and diluted with a cultured medium to 1 mg/ml. All cell lines with 3 × 10³ cells per well were cultured in a 96-well plate (SPL Life Sciences, Pochon, Kyonggi-do, Korea), after 24 h’ starvation, then treated with FK866, NAM, NMN in 1 % FBS medium for 48 h and incubated with a 1 mg/ml MTT solution for 2 h to obtain the crystal formazan. Subsequently, 100 μl DMSO was added to dissolve the formazan. The VERSA Max microplate reader (Molecular Devices, San Jose, CA, USA) was used at wavelengths 570 nm and 630 nm to measure the absorbance. The absorbance percentages were calculated by dividing them by the average value of the control.

2.10. Crystal violet staining

All cell lines were seeded in 12-well plate (5 × 10⁴ cells/well) (SPL life science), after 24 h’ starvation, then treated with FK866, NAM, NMN in 1 % FBS medium for 48 h. After the treatments, methanol (ECHO Chemical Co. Ltd) was used to fix the cells and they were stained with 0.5 % crystal violet (C3886, Sigma-Aldrich, St Louis, MO, USA)) for 15 min at room temperature. They were subsequently washed with PBS, air-dried and dissolved in DMSO (1 ml per well, ECHO Chemical Co. Ltd.), and 100 μL of each sample was transferred to 96-well plates and a wavelength of 550 nm was used to measure the absorbance by a microplate reader (Molecular Devices). The absorbance percentages were calculated by dividing them by the average value of the control, results were expressed as mean ± SD [25].

2.11. Oxidative stress evaluation

The cells were seeded in a 96-well plate at a density of 3000 cells per well. To assess oxidative stress, two reagents were used: H₂DCFDA (D399) and MitoSOX (M36008) (both from Thermo Fisher). H₂DCFDA, at a concentration of 20 μM, was used to evaluate overall ROS levels, while MitoSOX, at a concentration of 500 nM, was employed to specifically assess mitochondrial oxidative stress. After treating the cells and incubating them at 37 °C for 30 min, fluorescence was measured using a full-wavelength multifunctional microplate reader (Thermo Scientific Varioskan Flash Multimode Reader).

2.12. Colony formation

The cells were cultured in 6-well plate (500 cells/well), after 24 h’ starvation, then treated with FK866, NAM, NMN in 1 % FBS medium for 48 h. The medium was replaced with a 10 % FBS cultured medium for 7–10 days until the colony formed and was stained by 0.5 % crystal violet [14].

2.13. Western blot analysis

Cell (1 × 10⁵ cells) were seeded in 6 cm dish, after 24 h’ starvation, then treated with FK866, NAM, NMN in 1 % FBS medium for 48 h. The cellular and tissue samples were lysed with RIPA buffer-containing protease (4693132001) and phosphatase (4906837001) inhibitor (Roche, Basel, Switzerland), homogenized with LABSONIC® M and TissueLyser II (QIAGEN, Chatsworth, CA, USA), respectively, and centrifuged at 12,000 rpm for 30 min in 4 °C. Protein quantification was carried out with a BCA assay (T-Pro Biotechnology, New Taipei City, Taiwan). Used 20 μg (cell) or 80 μg (animal) an equal amount of protein loaded to each lane of 10–15 % SDS polyacrylamide gel electrophoresis separation, transfer to polyvinylidene fluoride membrane in 95V for 130 min (Millipore, Billerica, MA, USA) and being blocked by 5 % bovine serum albumin (BSA) for 1 h at room temperature. Primary antibodies, NAMPT (Proteintech Cat# 11776-1-AP, RRID:AB_2298317 (Rosemont, IL, USA)), Fibronectin (Abcam Cat# ab2413, RRID:AB_2262874), COL1A1 (GeneTex Cat# GTX112731S, RRID:AB_10727781 (Irvine, CA, USA)), α-SMA (GeneTex Cat# GTX100034, RRID:AB_1240408), MMP-9 (Santa Cruz Biotechnology Cat# sc-10737, RRID:AB_2266507 (Santa Cruz, CA, USA)), CathepsinB (Cell Signaling Technology Cat# 31718, RRID:AB_2687580 (Boston, MA, USA)), LC3B (Cell Signaling Technology Cat# 3868, RRID:AB_2137707), p62 (Cell Signaling Technology Cat# 39749, RRID:AB_2799160), ATG16L1 (Proteintech Cat# 67943-1-Ig, RRID:AB_2918695), PI3K (Cell Signaling Technology Cat# 4249, RRID:AB_2165248), Akt (Cell Signaling Technology Cat# 9272, RRID:AB_329827), p-Akt (Cell Signaling Technology Cat# 9271, RRID:AB_329825), PARP (Cell Signaling Technology Cat# 9542, RRID:AB_216073), Cyclin D1 (Abcam Cat# ab134175, RRID:AB_2750906), CD44 (Proteintech Cat# 15675-1-AP, RRID:AB_2076198), p-JNK (Cell Signaling Technology Cat# 9251, RRID:AB_331659), JNK (Cell Signaling Technology Cat# 9252, RRID:AB_2250373), OCT-4 (Proteintech Cat# 11263-1-AP, RRID:AB_2167545), IL-8 (Proteintech Cat# 27095-1-AP, RRID:AB_2861340), Transferrin (Santa Cruz Biotechnology Cat# sc-52256, RRID:AB_630356), GAPDH (Proteintech Cat# HRP-60004, RRID:AB_2737588) were diluted at 1:1000 by 5 % BSA and incubated overnight at 4 °C. After washing, the samples were incubated with horseradish peroxidase-conjugated goat anti-rabbit (111-035-003) or anti-mouse antibodies (115-035-003) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 2 h at room temperature. The chemical luminescence signal of protein expression was captured by an eBlot Touch Imager (eBlot Photoelectric Technology, Shanghai, China). ImageJ software (NIH, Bethesda, MD, USA) was used to measure the relative band. The density of the protein was normalized to GAPDH or transferrin (serum). The data expressed as fold change compared to the average value of untreated group (Control group), which was designed as 1.0 [26].

2.14. Immunofluorescence

The cells (5 × 10⁴ cells/well) were seeded on sterilized cover glasses (Paul Marienfeld, LaudaKönigshofen, Germany) in 6 well plate and after treatment were fixed with 4 % paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature, incubated for 5 min with 0.1 % Triton-X-100 for permeabilization and blocked by 5 % BSA for 30 min at room temperature. Subsequently, they were incubated with Fibronectin (ab2413, abcam), COL1A1 (GTX112731, Genetex), LC3B (3868, Cell signaling) or LAMP-1 (Proteintech Cat# 21997-1-AP, RRID: AB_2878966) (1:200 in 5 %BSA), followed by Alexa Fluor 546-goat anti-rabbit IgG antibody (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. The cells were mounted in EverBrite Hardset mounting medium with DAPI (Biotium, Fremont, CA) and images were captured by microscopy (Olympus (Tokyo, Japan)). The fluorescence signal was measured by Image J to calculate the intensity.

2.15. RT-PCR

RNA was isolated from ELT-3 cell by using TRIzol (TRI100, Bioman, Taiwan) and extract by Direct-zol RNA Miniprep kit (R2052, Zymo Research Corp., Irvine, CA, USA). RNA was reverse transcribed by using PrimeScript RT Reagent Kit (RR037B, TaKaRa, Shiga, Japan) and RT-qPCR was operated by QuantStudio-1 Real-Time PCR instrument (Thermo Fisher Scientific, Waltham, MA, USA) using SYBR Green Master Mix (Applied Biosystems no. 4367659). The following primer sequences were used for fn1 (Forward: 5′-GACTCGCTTTGACTTCACCAC-3′, Reverse: 5′-GCTGAGACCCAGGAGACCAC-3′), col1a1 (Forward: 5′- ATCAGCCCAAACCCCAAGGAGA-3′, Reverse: 5′-CGCAGGAAGGTCAGCTGGATAG-3′), and gapdh (Forward: 5′- CAGTCTTCTGGGTGGCAGTGAT-3′, Reverse: 5′- TGAGGCCGGTGCTGAGTATGT-3′). Sample were normalized using gapdh with delta delta CT approach for the quantitative analysis.

2.16. Next generation sequence sample preparation and establishment

The cells were seeded into 10-cm culture dishes. After treatment, all samples were collected using TRIzol reagent (Bioman, Taipei, Taiwan). RNA extraction and next-generation sequencing analysis were performed by Tursi Biotechnology Company. RNA purity was examined using the SimpliNano™ Biochrom Spectrophotometer (Biochrom, MA, USA), and further checked using the Qsep 100 DNA/RNA Analyzer (BiOptic Inc., Taiwan). Transcriptome sequencing libraries were prepared using the KAPA mRNA HyperPrep Kit (KAPA Biosystems, Roche, Basel, Switzerland), and the quality of the libraries was assessed using the Qubit@ 2.0 Fluorometer (Thermo Scientific) and the Agilent Bioanalyzer 2100 system.

2.17. RNA sequence data analysis

For gene expression difference was performed by DESeq2 (v 1.26.0) with biological duplicates. The p-values were adjusted using the Benjamini and Hochberg's method for controlling the false discovery rate (FDR). The gene set enrichment analysis (GSEA) was accomplished with 1000 permutations to analyze the enriched biological functions and activated signaling pathways from the molecular signature database (MSigDB). The MSigDB is a library used with the GSEA software, along with positional gene sets, hallmark gene sets, motif gene sets, curated gene sets, GO gene sets, computational gene sets, oncogenic gene sets and immunological gene sets.

2.18. Sphere formation assay

Modified from previous study [27]. The cells were seeded in 6-well plate (1 × 10⁵ cells/well). Following treatment, 200 cells per well were seeded in 96-well Ultra-Low Attachment Plates (3474, CORNING (Christiansburg, VA, USA)) and cultured in a medium supplied with 20 ng/ml epidermal growth factor (ALX-201-812-0100, Enzo life Sciences, Inc. (Farmingdale, NY, USA)), 10 ng/ml basic fibroblast growth factor (400-29, peprotech (Rocky Hill, NJ, USA)), 5 μg/ml insulin (Thermo), and 0.4 % Bovine Serum Albumin B-27(Gibco (Grand Island, NY, USA) for 14 days and the sphere diameter and number were calculated under microscopy.

2.19. Xenograft animal model

The animal studies were conducted under the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Taipei Medical University (No.: LAC-2019-0535). Five-week-old female balb/c nude mice were obtained from BioLASCO (Taipei, Taiwan) and maintained in a 12-h artificial illumination cycle and a temperature controlled room (22 ± 2 °C). The ELT-3-LUC (2 × 10⁶ cells/50 μl) was injected subcutaneously [23]. After 28 days of tumor growth, they were randomly divided into groups according to the tumor size. The FK866 (F10: 10 mg/kg [28]; F20: 20 mg/kg [29]) and the nicotinamide (N500: 500 mg/kg; N1500: 1500 mg/kg [30]) were injected intraperitoneally for 14 days. The NMN (300 mg/kg; 500 mg/kg [31]) was administered by intraperitoneal injection.

2.20. Serum NAMPT (extracellular NAMPT) evaluation

The NAMPT content in serum was determined using the Nampt (Visfatin/PBEF) (mouse/rat) Dual ELISA Kit (AG-45A-0007YEK-KI01, Adipogen International, San Diego, CA, USA) according to the instructions provided by the manufacturer. Briefly, the serum samples were incubated for overnight at 4 °C, and incubated with detection antibody for 1 h at 37 °C. After incubated with the diluted HRP Conjugated anti-guinea pig IgG (HRP) for 1 h at 37 °C and TMB substrate solution (TMB) for 10 min in room temperature to yield the blue solution. Added stop solution to turn to yellow and measure the wavelength at 450 nm.

2.21. NAD/NADH measurement

The NAD/NADH content in the tissues was determined with an Amplite® Colorimetric NAD/NADH Ratio Assay Kit (AAT Bioquest, Sunnyvale, CA, USA). A sample of 20 mg of tissue was extracted with the lysis buffer in the kit, and NAD and NADH were measured with the steps and reagents provided by the manufacturer. After incubating at room temperature for 60 min, the enzyme immunoassay analyzer was used for measurement at a wavelength of 460 nm.

2.22. Immunohistochemistry

The UL tissues were harvested, fixed in 10 % formalin, and embedded in paraffin for histological staining. Tissue sections (5 μm thick) were stained with Masson's trichrome, collagen type I alpha 1 (COL1A1), and nicotinamide phosphoribosyltransferase (NAMPT). The staining procedures were conducted by Bio-Check Laboratories Ltd. (Taipei, Taiwan). Microscopy (Olympus, Tokyo, Japan) was utilized for image capture, and ImageJ software was employed for calculating the positive area.

2.23. Statistical analysis

All data are presented as the mean ± SD (in vitro) or the mean ± SEM (in vivo). The correlation analysis from the tissue array and the GEO database was performed by Pearson's r correlation. A one-way ANOVA (analysis of variance) accompanied with Tukey's multiple comparisons test as a post hoc test was used to test the statistical difference between each group; p-values less than 0.05 were considered statistically significant. All analyses were performed using GraphPad Prism 9.0.

3. Results

3.1. NAMPT expression and the correlation analysis of NAMPT and COL1A1 in human uterine leiomyoma tissue

To investigate the involvement of NAMPT in UL progression, we examined the expression of NAMPT protein in paired human UL and myometrium tissues. The protein expression of NAMPT was found to be significantly higher (p < 0.01) and positively correlated with COL1A1 in UL compared to myometrium tissues, as depicted in Fig. 1A. This observation suggests that UL, characterized by enhanced proliferative capacity, relies on excessive utilization of NAMPT. To further assess the association between NAMPT and UL progression, we investigated the correlation between NAMPT expression and ECM-related proteins. Specifically, we examined the relationship between NAMPT and the accumulation of extracellular matrix protein COL1A1 using an SO806 tissue array chip. The results revealed a higher incidence of UL in women aged 32–49, and their histological sections displayed a positive correlation between the expression of COL1A1 and NAMPT (R = 0.3378, p = 0.0330), as depicted in Fig. 1B. These findings suggest that NAMPT potentially modulates the progression of UL by influencing the expression of COL1A1. Analysis of the Gene Expression Omnibus (GEO) database (GSE31699) demonstrated a higher risk of UL among African American women. Additionally, GEO database revealed the elevated levels of NAMPT in UL tissues (Fig. 1C, p < 0.05), with clinical characteristics further demonstrated a significant positive correlation between NAMPT levels and tumor weight (Fig. 1D (R = 0.6978, p = 0.0116)) and UL number (Fig. 1E (R = 0.6474, p = 0.0229)). Positive correlations observed between COL1A1 and NAMPT (Fig. 1F (R = 0.5321, p = 0.0339)), as well as between MMP-9 and NAMPT (Fig. 1G (R = 0.6224, p = 0.0100)) and the stemness marker CD44 (Fig. 1H (R = 0.7895, p = 0.0008)). These results indicate that NAMPT may regulate the progression of ECM. Taken together, these findings indicate that NAMPT is associated with UL progression. Consequently, our objective is to investigate the modulation of UL progression through NAMPT inhibition.

Fig. 1.

NAMPT expression and correlation with COL1A1 in leiomyoma tissue.

(A) NAMPT and COL1A1 expression in UL tissue (N = 9 cases). M, myometrium; L, uterine leiomyoma. (B) SO806 tissue microarray were stained with COL1A1 or NAMPT. The correlation between COL1A1 and NAMPT. Age range from 32 to 49, N = 40 cases. Used GEO database GSE31699 to determine (C) NAMPT mRNA level (N = 9) between M and L, Data represent the means ± SD. ∗, p < 0.05; ∗∗, p < 0.01 compared with control group. The correlation of (D) tumor size and NAMPT (N = 12), (E) tumor number and NAMPT (N = 12), (F) COL1A1 and NAMPT (N = 16), (G) MMP-9 and NAMPT (N = 16) and (H) CD44 and NAMPT (N = 14). P value was calculated using Pearson's correlation.

3.2. Role of NAMPT on UL progression

Subsequently, we utilized a human leiomyoma (HuLM) cell line to establish a NAMPT knockdown model, aiming to elucidate the central role of NAMPT. Following successful NAMPT silencing, a striking reduction in NAMPT protein expression was observed. Moreover, the expression of key extracellular matrix (ECM) components, fibronectin and COL1A1, was significantly downregulated upon NAMPT modulation (Fig. 2A). Notably, there was a substantial decrease in COL1A1 fluorescence following NAMPT knockdown (Fig. 2B), confirming the positive correlation previously demonstrated in Fig. 1B between NAMPT and COL1A1 expression. Likewise, siNAMPT-mediated downregulation of NAMPT protein expression had a profound impact on the regulation of the extracellular matrix, as depicted in the Volcano plot (Fig. 2C). GSEA analysis further underscores the significant modulation of ECM-associated gene sets resulting from NAMPT inhibition, with a marked decrease in their expression (Fig. 2D) and UL progression related gene (Fig. 2E). These findings underscore the critical influence of NAMPT regulation on the extracellular matrix. The attenuation of NAMPT function results in a notable decrease in ECM-related protein expression, providing robust evidence of NAMPT's pivotal role in effectively regulating ECM accumulation.

Fig. 2.

Role of NAMPT on UL progression.

HuLM cells (2 × 10⁵ cells/well) were cultured in 6-well plates for 24 h. Lipofectamine 3000 (Thermo Fisher Scientific) transfect different amount of ON-TARGET plus siNAMPT or non-targeting (Nt) sequence (nM) (Dharmacon) in Opi-MEM for 48 h. Protein expression of (A) NAMPT, fibronectin and COL1A1 were measured by Western blot (n = 3–4). (B) Representative image of COL1A1 staining in siNAMPT treatment. In 100× magnification, scale bar: 200 μm. (C) A volcano plot illustrates the gene expression changes in HuLM cells after siNAMPT treatment. The x-axis represents the scaled concordance value, with values above 0 indicating a positive association and values below 0 indicating a negative association. The y-axis shows the corresponding -log10 FDR-adjusted P-value. (D) siNAMPT GSEA enrichment analysis on extracellular matrix and (E) Heatmap of log2 fold changes of genes associated with UL progression between HuLM and UtSMC or after NAMPT inhibitor or siNAMPT treatment in HuLM. Rows are differentially expressed genes (DEGs) following RNA sequencing. Red denotes higher concentration or larger changes in mRNA concentration whereas blue denotes the opposite. Data represent the means ± SD,∗, p < 0.05; ∗∗∗, p < 0.001 compared with control group, used one-way ANOVA followed by a Tukey post hoc test for multigroup comparison. Scale bar: 200 μm.

3.3. Regulation of NAMPT enzyme activity and protein expression by FK866 and NAM

To assesses the impact of NAMPT inhibition, we evaluated both NAMPT enzyme activity and protein expression in ELT3 cells, a rat uterine leiomyoma (UL) cell line, following treatment with the NAMPT inhibitor FK866 and its metabolite NAM. FK866, a well-established specific NAMPT inhibitor, demonstrated a marked reduction in NAMPT activity. Additionally, NAM, a precursor of NAMPT, has been reported to inhibit eNAMPT [32]. To gauge the inhibitory potential of these NAMPT inhibitors, we conducted analyses of enzyme activity and protein expression. The results indicated that FK866 at concentrations ranging from 100 to 1000 nM exhibited a significant inhibitory effect on NAMPT activity (Fig. 3A), without affecting its protein expression (Fig. 3B). Conversely, a lower dose of NAM (5–20 mM) effectively inhibited NAMPT enzyme activity (Fig. 3A), while a higher dose (50 mM) significantly suppressed NAMPT protein expression (Fig. 3B). These findings underscore the regulatory role of FK866 and NAM in modulating NAMPT in the context of UL.

Fig. 3.

Effect of FK866 and NAM on the NAMPT regulation and proliferation modulation.

(A) NAMPT and NAD metabolism pathway, an NAMPT enzyme activity assay kit was performed. The positive control (PC) consisted of FK866 at 4 μM (n = 3). (B) NAMPT protein expression with ELT-3 cells were serum-starved for 24 h and then treated with FK866 and NAM for 48 h (n = 3). Cell viability was analyzed by (C) MTT assay, ELT-3 cells were treated with various concentrations of FK866 or NAM for 48 h (n = 5–6). (D) Crystal violet staining: Cell survival was assessed after treatment with (E) FK866 NAM in ELT-3 cells (1 × 10⁵ cells) cultured in a 6 well plate. (F) ROS levels were evaluated using H₂DCFDA and (G) mitochondria oxidative stress was assessed using MitoSOX. ELT-3 cells (1 × 10⁵ cells) were cultured in a 6 cm dish, treated with FK866 or NAM for 48 h (n = 3–5). (H) Protein expression of apoptosis and DNA damage related protein expresision and (I) cell cycle-related markers (PI3K, p-Akt/Akt ratio, cyclin D1) were evaluated by Western blot and quantified using Image J (n = 3–4). C, control; F, FK866; N, NAM. Data represent the means ± SD, ∗∗, p < 0.01; ∗∗∗, p < 0.001 compared with control group, used one-way ANOVA followed by a Tukey post hoc test for multigroup comparison.

3.4. Growth inhibitory effect of FK866 and NAM on the proliferation of ELT-3 cells

FK866 and NAM have previously exhibited anti-proliferative effects in cancer [14,33,34]; However, there has been a paucity of research regarding their impact on UL. Hence, our study represents the first investigation into the anti-proliferative effects of NAMPT inhibitors on cell viability. To assess the influence of NAMPT inhibitors on UL cells, FK866 (0.1–1000 nM) and NAM (1–50 mM) were administered for 48 h, and cell viabilities were determined via MTT assay. Notably, there was a significant dose-dependent inhibition of cell proliferation observed after 48 h of treatment with NAMPT inhibitors (Fig. 3C). These results were further corroborated using crystal violet staining, which yielded consistent findings with the MTT assay. Both FK866 and nicotinamide effectively curtailed the proliferation of ELT-3 cell lines, underscoring the potential of NAMPT inhibitors to impede the proliferative capacity of UL cells (Fig. 3D and E). Interestingly, in the myometrium, FK866 did not induce significant changes in cell viability (Supplementary Figs. 1A and C). Conversely, NAM demonstrated more pronounced inhibitory effects on UL cells (Supplementary Figs. 1B and D). Additionally, the NAD precursor NMN was found to activate NAD [35] and enhance cell viability in UL (Supplementary Fig. 2), further implicating the involvement of NAMPT and NAD in modulating the proliferative potential of UL cells.

3.5. The effect of NAMPT inhibitor on the proliferation-related protein expression regulation

To elucidate the underlying mechanisms of NAMPT's role in proliferative modulation, treatment with the NAMPT inhibitor resulted in elevated oxidative stress (Fig. 3F) and mitochondrial oxidative stress (Fig. 3G). This increase in oxidative stress may lead to heightened apoptosis and DNA damage (Fig. 3H), which, in turn, significantly reduces the protein expression of PI3K/Akt and cyclin D1 (Fig. 3I). These findings suggest that FK866 and NAM inhibit the activation of the PI3K/Akt pathway and downregulate cyclin D1, a crucial regulator of cell cycle progression. Thus, NAMPT inhibitors could be a promising therapeutic strategy for controlling the proliferation of UL cells.

3.6. The regulatory effect of FK866 and NAM on extracellular matrix accumulation in uterine leiomyoma

The accumulation of extracellular matrix (ECM) plays a pivotal role in the progression of UL, and targeting ECM accumulation has emerged as a crucial therapeutic approach for managing UL [36]. Although FK866 and NAM have demonstrated their anti-fibrotic properties, their specific effects on inhibiting ECM accumulation in UL have not been extensively explored. To assess the inhibitory influence of NAMPT on ECM accumulation, we conducted Western blot analyses to evaluate the expression of ECM-related proteins, including fibronectin, COL1A1, and α-SMA, in ELT3 cells treated with FK866 and NAM. The results unequivocally demonstrated that both treatments significantly reduced the immunofluorescence signal and the expression of all ECM-related proteins (Fig. 4A–C,D). Additionally, we conducted analyses of ECM-related protein expression, as shown in Supplementary Fig. 3, which was also observed in a human uterine leiomyoma cell line. This compelling evidence suggests that NAMPT inhibition can effectively suppress ECM accumulation in UL cells, offering potential therapeutic implications for modulating ECM accumulation in UL. Moreover, the immunoprecipitation was conducted to confirm the interaction between α-SMA and NAMPT (Fig. 4B) To investigate the regulatory role of NAMPT on ECM gene sets, we performed Gene Set Enrichment Analysis (GSEA) to scrutinize the expression of genes associated with extracellular matrix-related pathways. The analysis revealed that both FK866 and NAM interventions notably inhibited and regulated the expression of extracellular matrix-related gene sets (Fig. 4E and F) and ECM related genes level (Fig. 4G and H) in rat and human UL cells (Fig. 4I and J). These findings emphasize the crucial role of genetic regulation in the pathogenesis of UL and unveil potential therapeutic targets for managing its progression. It is worth noting that NMN, a precursor of NAD, significantly increased the expression of ECM-related proteins (Supplementary Fig. 4), suggesting a potential role of NAMPT and NAD in promoting ECM accumulation in UL. Considering that current clinical treatments for UL primarily focus on the anti-proliferative process and do not specifically target the anti-fibrotic process [36], the ability of NAMPT inhibitors to reduce ECM expression represents a critical modulation of UL progression. By inhibiting ECM accumulation, NAMPT inhibitors offer a promising avenue for the development of therapeutic strategies targeting UL-associated fibrosis.

Fig. 4.

Effect of FK866 and NAM on extracellular matrix accumulation.

ELT-3 cells (1 × 10⁵ cells) were cultured in 6 cm dish, cells were serum starved for 24 h, were exposed to varying concentrations of FK866 (ranging from 100 to 1000 nM) or NAM (ranging from 5 to 50 mM) for a duration of 48 h. (A) Immunocytochemistry was used to measure the fluorescence of Fibronectin and COL1A1 (n = 4–5). (B) Interaction between α-SMA and NAMPT by immunoprecipitation (n = 3). (C,D) Western blot analysis was employed to assess the modulation of FK866 and NAM on ECM-related protein expression (n = 4–5). (E) GSEA enrichment analysis (depicted in F1000) highlights the impact of FK866 (1000 nM) treatment, while (F) N50 represents NAM (50 mM) treatment, on the extracellular matrix-related gene set in ELT-3 cells. (G) Gene levels of col1a1 and fn1 after (G) FK866 treatment (n = 3–9). and (H) NAM treatment = (n = 3–9). GSEA enrichment analysis conducted on HuLM cells after (I) FK866 treatment and (J) NAM treatment. Data represent the means ± SD, ∗∗, p < 0.01; ∗∗∗, p < 0.001 compared with control group, used one-way ANOVA followed by a Tukey post hoc test for multigroup comparison.

In a previous study, the significance of lysosome function in depleting ECM accumulation was elucidated [37]. In our investigation, we observed an increase in lysosome immunofluorescence signal following treatment with the NAMPT inhibitor (Supplementary Fig. 5A). Specifically, modulation of cathepsin B, known for its role in ECM degradation, was noted [38]. To induce cathepsin B dysfunction, we utilized Bafilomycin A1 (BaFA1), resulting in a significant reduction of the active form of cathepsin B. This led to an upsurge in the expression of ECM-related proteins and halted the function of the NAMPT inhibitor in reducing ECM levels (Supplementary Figs. 5B and C).

The restoration of autophagy activity has been shown to mitigate fibrosis progression [39]. Activation of autophagy influx can increase the expression of COL1A1 [40]. In our study, we delved further into the effect of NAMPT inhibitors on autophagy influx. Both FK866 and NAM inhibited the formation of LC3BII, accompanied by a decrease in p62 and ATG16L1 protein expression (Supplementary Figs. 6A and B). To investigate the role of phagophore formation, we examined its progression and the function of ATG16L1. Depletion of ATG16L1 impaired LC3BII formation and autophagy influx. The involvement of phagophore formation in ECM modulation was confirmed by using 3-MA (a phagophore inhibitor) and Rapamycin (a phagophore formation inducer). Inhibition of phagophore formation strengthened the modulation by NAMPT inhibitors, while activation restored their abilities (Supplementary Figs. 6C–E). The NAMPT inhibitor demonstrated a reduction in autophagy, which may contribute to its role in modulating ECM accumulation.

3.7. Suppression of Stemness Following NAMPT inhibition

To investigate the phenomenon of stemness in UL, we conducted a sphere formation assay and colony test. Our results unequivocally demonstrated that both FK866 and NAM exhibited significant inhibitory effects on both the diameter and number of formed spheres (Fig. 5A and B) as well as the number of colonies formed (Fig. 5C and D). The interventions with FK866 and NAM effectively suppressed the expression of the stem cell marker CD44 and the stemness-related protein OCT-4. Additionally, the stemness phenotype and the expression of the ECM transcription signaling protein p-JNK/JNK were decreased, highlighting the regulatory effects of FK866 and NAM on the stemness and ECM of UL (Fig. 5E and F). It's noteworthy that NMN, a precursor of NAD, resulted in an increased diameter and number of sphere formations, along with the induction of stemness-related protein expression (Supplementary Fig. 7). This observation suggests that UL formation may indeed originate from the stemness of myometrium and create a conducive environment for tumorigenesis. In summary, our findings suggest that NAMPT inhibitors can effectively retain the self-renewal ability of UL cells and modulate signaling pathways involved in stemness and ECM formation.

Fig. 5.

Suppression of Stemness Following NAMPT Inhibition.

ELT-3 cells (1 × 10⁵ cells/well) were cultured in 6-well plates, cells were serum free starvation for 24H and treated with various concentrations of FK866 or NAM for 48 h (A)Sphere formation was performed and microscopy was employed for image capture. (B) the sphere diameter and sphere numbers were quantified (n = 7–10). (C) colony formation was captured, and (D) crystal violet staining was applied to visualize and quantify the colonies (n = 4). (E) stemness protein epxression was examined by Western blot. (F) Western blot analysis was performed (n = 3–4). Data are presented as means ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 compared with the control group. Statistical analysis was conducted using one-way ANOVA followed by a Tukey post hoc test for multiple group comparisons.

3.8. Assessing Therapeutic Potential of NAMPT Inhibitors In Vivo

To evaluate the therapeutic potential of NAMPT inhibitors, we employed a xenograft animal model. FK866 and NAM, NAMPT inhibitors, were administered via intraperitoneal injection. Notably, after just 14 days of treatment, both FK866 and NAM interventions significantly reduced both the size and weight of the tumor tissue, indicating effective inhibition of UL proliferation (Fig. 6A–D). In stark contrast, NMN treatment led to the activation of UL proliferation (Supplementary Figs. 8A–C), reinforcing the critical role of NAMPT in modulating tumor growth. Moreover, we assessed the protein expression of ECM-related markers, including fibronectin, COL1A1, and α-SMA, within the xenograft tumors. Treatment with FK866 and NAM resulted in a substantial decrease in the protein expression of these ECM markers (Fig. 6E–G), providing further confirmation of the pivotal role of NAMPT in regulating ECM accumulation. Conversely, NMN treatment increased the expression of ECM-related proteins (Supplementary Figs. 8D and E), indicating a pro-fibrotic effect. These in vivo findings serve as additional compelling evidence supporting the involvement of NAMPT in the regulation of tumor cell proliferation and ECM accumulation, consistent with observations made in our in vitro models.

Fig. 6.

Assessing Therapeutic Potential of NAMPT Inhibitors In Vivo.

ELT-3-LUC cells (2 × 10⁶ cells) were subcutaneously implanted into the right lateral abdomen of nude mice. After 28 days of implantation, mice were treated with FK866 at doses of 10 mg/kg (F10, i.p.) or 20 mg/kg (F20, i.p.) or with Nicotinamide (NAM) at doses of 500 mg/kg (N500, i.p.) or 1500 mg/kg (N1500, i.p.) once daily. Tumor size and weight were measured after 14 days of treatment. (A) The flowchart of the intervention: After the treatment, the tumor size and weight were measured. (B) Changes in tumor size over the course of the treatment (n = 4–5). (C) Representative images of tumors in the FK866-treated and NAM-treated groups. (D) ECM-related protein expressions were examined by Western blot. (E) Western blot analysis was performed to assess the expression of extracellular matrix-related proteins in tumors from the FK866 and NAM-treated groups (n = 3–4). Data are presented as means ± SEM. Significance levels are denoted as follows: ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001 compared with the control group, denoted as "C." Statistical analysis was carried out using one-way ANOVA followed by a Tukey post hoc test for multiple group comparisons.

3.9. The effects of the NAMPT inhibitor on NAMPT and IL-8 secretion and NAD metabolism

To gain deeper insights into the regulation of NAMPT and its implications in tumor suppression, we conducted additional analyses. Specifically, we investigated protein expression in the serum and observed that treatment with FK866 and NAM led to a significant inhibition of eNAMPT and IL-8 secretion (Fig. 7A–C). These findings suggest that NAMPT inhibitors can modulate the tumor microenvironment by suppressing the secretion of eNAMPT and IL-8. Conversely, the activator NMN influenced NAD metabolism, resulting in increased NAMPT and IL-8 secretion (Supplementary Fig. 8F-I).

Fig. 7.

Effects of NAMPT Inhibition on Extracellular NAMPT, IL-8, NAD Levels, and ECM Interaction

(A,B) Extracellular NAMPT and IL-8 expression were assessed in the serum of FK866-treated and NAM-treated groups using Western blot (n = 4). (C) ELISA was employed to measure extracellular NAMPT levels (n = 4). (D) NAD/NADH and NAD⁺ levels in tumors from the FK866-treated and NAM-treated groups were measured (n = 4). (E) Tumor immunohistology was performed and stained for NAMPT, COL1A1 and Masson trichrome, with a scale bar of 50 μm. (F) FK866 and (G) NAM groups quantifications were done by image J (n = 6–10). (H) The correlation between COL1A1 and NAMPT or Masson trichrome and NAMPT staining areas were used to illustrate the interaction of NAMPT in extracellular matrix (ECM) modulation (n = 19). The data are presented as means ± SEM, and significance levels are indicated as follows: ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001, compared with the control group, denoted as "C." Statistical analysis was performed using one-way ANOVA followed by a Tukey post hoc test for multiple group comparisons.

Moreover, treatment with FK866 and NAM significantly inhibited NAD metabolism (Fig. 7D), suggesting its potential impact on extracellular NAMPT protein expression. This decrease in NAD metabolism correlates with reduced proliferation and tumorigenicity. We utilized immunohistochemistry (IHC) to evaluate the effect of NAMPT inhibition on NAMPT and COL1A1 modulation. The IHC analysis revealed a notable decrease in NAMPT and COL1A1 expression (Fig. 7E–G). Masson's trichrome staining indicated the positive area of ECM accumulation, with FK866 and NAM treatment showing a reduction in the positive area, which positively correlated with NAMPT levels observed in the in vivo model (Fig. 7H). Overall, our results underscore the critical role of NAMPT in suppressing UL progression by influencing its microenvironment. This effect is accompanied by a decrease in ECM-related protein expression and a depletion of NAD, effectively modulating the progression of UL.

4. Discussion

The findings of this study shed light on the regulatory role of NAMPT in UL proliferation, ECM accumulation, and stemness progression. It is indeed the first study to establish the correlation between NAMPT and ECM accumulation, highlighting the potential therapeutic implications of NAMPT inhibitors in the management of UL.

Previous studies have implicated that a reduction in NAD⁺ levels can lead to the activation of reactive oxygen species (ROS) [41], which may accelerate PARP cleavage independent of caspase-3 activation, thereby modulating DNA fragmentation and ultimately causing cell death [42]. In our results, treatment with the NAMPT inhibitor led to an elevation of both ROS and mitochondrial ROS, alongside the induction of cleaved PARP and γ-H2AX protein expression. Notably, the activation of cleaved PARP was accompanied by an increase in PARP-1, a known DNA damage sensor, which can promote DNA fragmentation and trigger cell death [43]. These findings suggest that the observed increase in γ-H2AX, along with the induction of Bax/Bcl2 and cleaved PARP, is associated with elevated ROS levels following NAMPT inhibitor treatment.

In the context of UL pathogenesis, the PI3K/Akt pathway is frequently induced and serves as one of the key signaling pathways in UL. It regulates the response to estrogen induction and has been identified as a potential target for regulation. However, clinical trials targeting the PI3K/Akt/mTOR pathway have shown severe side effects, including hot flashes, fatigue, and diarrhea [44]. On the other hand, NAMPT activation promotes the cancer cell cycle by activating cyclin D1 and increasing DNA synthesis, leading to enhanced cancer cell proliferation [6,14]. Moreover, NAMPT creates a supportive environment for angiogenesis and cancer proliferation [45]. UL cells are characterized by a high proliferation rate and elevated expression of cyclin D1 [46]. Our current findings demonstrate that NAMPT inhibitor can decrease the activation of the PI3K/Akt pathway, induce cell cycle arrest, and promote apoptosis, thus suppressing UL proliferation.

UL is a monoclonal tumor that originates from the transformation of myometrium stem cells into leiomyoma stem cells, which undergo self-renewal through division, increasing their proliferative capacity and contributing to tumor formation [7,47]. Additionally, these cells exert paracrine effects, enhancing the sensitivity of surrounding tissues to estrogen and progesterone, thereby inducing related signaling pathways that promote further proliferation of UL [8]. Leiomyoma stem cells are characterized by higher expression of stem cell markers such as OCT-4 and CD44 [47]. They also activate signaling pathways such as Wnt/β-catenin and c-JUN N-terminal kinase (JNK), which stimulate cell proliferation and increase extracellular matrix (ECM) accumulation. JNK signaling can regulate the transactivation of MMP-9, which may be involved in the inhibition of ECM accumulation [48]. Stem cells exhibit energy utilization patterns resembling the Warburg effect, relying on NAD as a coenzyme and energy source [49]. NAMPT, a key enzyme in NAD metabolism, is highly expressed in stem cells and plays a role in regulating autophagy and stem cell formation through OCT-4 modulation [10]. Furthermore, NAMPT enhances DNA repair and autophagy, contributing to stem cell development through its self-renewal capacity. Our findings suggest that NAMPT mRNA level positively correlated to CD44 stemness related gene expression and the inhibition of NAMPT decreases sphere formation and the expression of stemness-related markers, indicating a reduction in self-renewal ability, including the decrease in CD44 expression. This may explain the regulatory role of NAMPT inhibition in modulating markers such as OCT-4, JNK, MAPK signaling, and ECM accumulation.

Autophagy as one of the major modulator in ECM accumulation [50]. The overexpression of NAMPT has also shown autophagy induction through an increase in the LC3BII and beclin1 protein expression [51]. In transforming growth factor-β1 (TGF-β1)-induced fibrogenesis, the LC3BII accumulation and p62 degradation is accompanied by ECM accumulation [52]. In autophagy initiation, ATG16L1 plays a key role in the autophagy elongation leading to phagophore formation [53]. As ATG16L1 leads the LC3B lipidation, therefore the alteration of ATG16L1 would inhibit the ATG12-ATG5 localization and the autophagosome formation causing autophagy interruption. The autophagy influx and cytokine secretion have been related to cancer tumorigenesis. The co-culturing of fibroblasts and breast cancer cells activated the IL-8 and IL-10 secretion and induced the autophagy influx [54].

The role of NAMPT in NAD homeostasis is still not fully understood and remains controversial. eNAMPT has been implicated as a cytokine or adipokine and is involved in maintaining NAD metabolism [32]. In vivo study presented here demonstrates that inhibition of eNAMPT results in the absence of circulating NAMPT, decreased NAD metabolism, potentially influencing NAD utilization in hyper-proliferative tissue and resulting in a smaller tumor size and reduced IL-8 secretion. These findings suggest that NAD utilization and NAMPT modulation play important roles in UL progression.

On the other hand, NMN, as an NAD precursor and a product of NAMPT, can boost the NAD pool and NAD metabolism, providing anti-aging effects and improving insulin sensitivity, which may have therapeutic potential in metabolic disorders [55]. However, NAD boosting may also increase energy metabolism and contribute to carcinogenesis. While NMN supplementation offers several benefits, our results indicate that in UL, which exhibits high NAMPT utilization, the use of NAD booster supplements should be carefully considered in patients.

In conclusion, our results provide evidence for the involvement of NAMPT in the progression of UL and highlight its potential as a therapeutic target. The findings demonstrate that NAMPT expression is significantly higher in UL tissues compared to normal myometrium tissues, suggesting its role in UL progression. The results further support the notion that NAMPT inhibitors, such as FK866 and NAM, can effectively inhibit NAMPT enzyme activity and suppress UL cell proliferation. These inhibitors show promising effects in reducing ECM accumulation in UL cells, suggesting their potential as anti-fibrotic agents. Furthermore, our results reveal the influence of NAMPT on NAD metabolism, eNAMPT and IL-8 secretion, and the tumor microenvironment, suggesting its involvement in modulating the UL tumor microenvironment (Fig. 8). Overall, these findings contribute to the understanding of the role of NAMPT in UL progression and provide a basis for further research on the development of therapeutic strategies targeting NAMPT for the treatment of UL.

Fig. 8.

NAMPT role in uterine leiomyoma progression

Schematic representation illustrating the role of nicotinamide phosphoribosyltransferase (NAMPT) in the progression of uterine leiomyoma (UL). The positive correlation between NAMPT expression and markers of ECM and stemness. It also depicts the mechanisms by which NAMPT inhibition, using inhibitors such as FK866 and NAM, reduces UL cell survival, disrupts autophagy, and maintains lysosomal function homeostasis, thereby preventing further ECM accumulation.

CRediT authorship contribution statement

Yi-Fen Chiang: Writing – original draft, Investigation, Conceptualization. Ko-Chieh Huang: Methodology, Investigation. Tsui-Chin Huang: Resources, Methodology. Hsin-Yuan Chen: Methodology. Mohamed Ali: Resources, Methodology. Ayman Al-Hendy: Resources. Pei-Shen Huang: Resources. Shih-Min Hsia: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

Compliance with ethics requirements

The human study was approved by the Institutional Review Board and Ethics Committee of Taipei Medical University Hospital (Permit Number: N202305018).

The animal studies were conducted according to the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Taipei Medical University (No.: LAC-2019-0535).

Data sharing statement

To request access, please contact the lead contact. Requests will be answered within 4 weeks. Source data supporting the findings of this study are available within the article and Supplementary information files.

Declaration of competing interest

The authors have declared no conflict of interest.

Appendix A. Supplementary data

The following are the Supplementary data to this article.

Data availability

Data will be made available on request.