Association between different levels of lipid metabolism‑related enzymes and fatty acid synthase in Wilms' tumor
- Authors:
- Published online on: December 20, 2019 https://doi.org/10.3892/ijo.2019.4948
- Pages: 568-580
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Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Wilms’ tumor is one of the most common malignant tumors of the abdomen in children. The prognosis of this cancer is associated with age, tumor size and histological type. However, the most important prognostic factor is local invasion and distant metastasis of the tumor (1). The mechanism underlying the pathogenesis and metastasis of Wilms’ tumor have yet to be fully elucidated, and further research is required to design targeted therapies.
The role of lipid metabolism in cancer is attracting increasing attention, and inhibition of lipid autophagy has been shown to be of clinical value in other tumors (2). Due to the high metabolic demands of tumor cells and their nutritional deficiency during chemoradiotherapy, fatty acid metabolism can provide the necessary energy for tumor cells. Furthermore, tumor cell division requires a high energy supply. As phospho-lipids are an important part of the cytoskeleton, phospholipid synthesis provides essential raw materials for tumor cell proliferation (3-5). Thus, recent publications have reported that lipid metabolism is closely associated with tumor cell proliferation, invasion, migration and chemosensitivity (6,7), and it is emerging as an attractive target for cancer drug discovery.
Fatty acid synthase (FASN) is essential for de novo lipo-genesis and cellular substrate energy metabolism, including the synthesis of long-chain fatty acids from acetyl-CoA, malonyl-CoA and NADPH (8,9). The expression of FASN also has a considerable impact on tumor progression: The expression of FASN in a variety of solid tumors was found to be significantly increased and was associated with poor prognosis (10), and the proliferation, invasion and migration of tumor cells were significantly suppressed after its downregulation (11-13). In particular, in renal cell carcinoma, Albigeset al demonstrated that FASN played an important role in the occurrence of tumors and that patients with higher expression levels had a poor prognosis (14). However, only few studies (15) have addressed the role of FASN in Wilms’ tumor.
Investigating the effect and mechanism of action of FASN, a lipid-metabolizing protein, on the metastasis of Wilms’ tumors may provide a novel approach to the targeted therapy of this disease. The aim of the present study was to examine the different levels of lipid metabolism-related enzymes and determine whether FASN is a key factor in the progression of Wilms’ tumor.
Materials and methods
Tissues
Between January 2007 and January 2012, a total of 65 patients with Wilms’ tumor who did not undergo radical or palliative nephrectomy were selected for immunohistochemistry (IHC) analysis. Clinical data, including sex, age, tumor size, stage, histopathological type, metastasis and follow-up information, were recorded. The patients included 35 males and 30 females, with a mean age at diagnosis of 3.2 years (range, 0.25-11.8 years). Detailed information may be found in Table I. The duration of the follow-up was 60 months. In addition, 20 cases of Wilms’ tumor and adjacent tissues collected between March 2015 and December 2017 and stored at -80°C were selected. The tumors were graded according to the Fuhrman nuclear grade classification (16) as follows: G1, n=10; G2, n=7; and G3, n=3. A total of 9 cases (G1, n=3; G2, n=3; and G3, n=3) were used for label-free quantification. Radiotherapy, chemotherapy and immunotherapy were not performed prior to surgery, and the samples were verified by two pathologists postoperatively. The study protocol was approved by the Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong University, and the legal guardians of the patients provided informed consent regarding the use of the patients’ tissues for research purposes.
 | Table ICorrelation of expression levels of the FASN protein with the clinicopathological parameters of patients with Wilms’ tumor. |
Protein digestion
Proteins were re-dissolved in 500 mM triethylammonium bicarbonate (TEAB). The protein concentration of the supernatant was determined using the BCA protein assay, and 100 µg protein per condition was transferred into a new tube and adjusted to a final volume of 100 µl with 8 M urea. Subsequently, 11 µl of 1 M DTT was added, and the sample was incubated at 37°C for 1 h and transferred into a 10 K ultrafiltration tube (EMD Millipore). To remove the urea, the samples were centrifuged after adding 100 mM TEAB three times at 14,000 × g at 4°C for 10 min, followed by the addition of 120 µl of 55 mM iodoacetamide to the sample and incubation for 20 min in the dark at room temperature. The proteins were then digested with sequencing grade modified trypsin (Promega Corporation).
Label-free quantification
First, 18 cases of in-solution digestion samples (Wilms’ tumor and their adjacent tissues; G1, n=3; G2, n=3; and G3, n=3) were acquired and analyzed by nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). The samples were resuspended in 30 µl solvent C (C: water with 0.1% formic acid; D: ACN with 0.1% formic acid), separated by nanoLC and analyzed by online electrospray MS/MS. The experiments were performed on an Easy-nLC 1000 system (Thermo Fisher Scientific, Inc.) connected to a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Inc.) equipped with an online nano-electrospray ion source. A 10-µl peptide sample was loaded onto the trap column (Acclaim PepMap C18, 100 µm × 2 cm, Thermo Fisher Scientific, Inc.) at a flow rate of 10 µl/min for 3 min, and subsequently separated on the analytical column (Acclaim PepMap C18, 75 µm × 15 cm) with a linear gradient from 3% D to 32% D in 60 min. The column was re-equilibrated at the initial conditions for 10 min. The column flow rate was maintained at 300 nl/min. The electrospray voltage of 2 kV vs. the inlet of the mass spectrometer was used.
The mass spectrometer was run under data-dependent acquisition mode and automatically switched under MS and MS/MS mode. The MS1 mass resolution was set as 35 K with m/z 350-1,550, and the MS/MS resolution was set as 17.5 K under HCD mode. The dynamic exclusion time was set at 20 sec.
Volcano plot
The volcano plot, which plots significance vs. fold-change on the y and x axes, respectively, is a type of scatter plot that is used to quickly identify changes in large datasets composed of replicate data. This plot is drawn by using the ggplot2 package (http://ggplot2.org).
Hierarchical cluster analysis (HCA)
HCA is an algorithmic approach to finding discrete groups with varying degrees of (dis)similarity in a dataset represented by a (dis)similarity matrix. This analysis is processed with the pheatmap package (https://CRAN.R-project.org/package=pheatmap).
Gene Ontology (GO) analysis
Blast2GO version 4 (BioBam Bioinformatics S.L.) was used for functional annotation. The whole protein sequence database was analyzed by BlastP using a whole database and mapped and annotated with a GO database. Statistically altered functions of differentially expressed proteins were calculated by Fisher’s exact test in Blast2GO.
Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis
The protein-protein interaction network was analyzed using STRING v.10 (http://string-db.org/).
Database for Annotation, Visualization and Integrated Discovery (DAVID) functional annotation tool
The differentially expressed proteins were also identified using DAVID functional annotation for functional analysis (https://david.ncifcrf.gov/).
Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis
Pathway analysis was processed by KOBAS (http://kobas.cbi.pku.edu.cn/). Pathways with P<0.05 were recognized as significantly altered.
Ingenuity Pathway Analysis (IPA)
IPA is a common tool used to model, analyze and understand complex biological systems, particularly to reveal different signaling networks and biological processes (17). IPA is an integrated biological pathway analysis software based on cloud computing. With the support of the highly structured bioinformatics platform Ingenuity Knowledge Base, it can search for various types of related information on genes, proteins and drugs, as well as constructing interaction models. In addition, this tool can also analyze data obtained through such techniques as genomics, microRNA, experimental data for single-nucleotide polymorphisms, chips, metabolomes and proteomes.
Immunohistochemistry analysis
Tissues obtained from Wilms’ tumors were subjected to fixation with 4% paraformaldehyde for at least 72 h at room temperature, dehydration using 10% formalin for 3 h, followed by immersion in a series of graded ethanol baths, washing in xylene, immersion and final embedding in paraffin. The tissue blocks were cut into 5-µm sections. After dewaxing and rehydration, heat-induced epitope retrieval was performed with 1 mM EDTA (pH 9.0) in a microwave oven. Then, samples were immersed in 3% H2O2 aqueous solution for 30 min to exhaust endogenous peroxidase. After closure blocking with goat serum for 30 min, the sections were incubated overnight at 4°C with primary anti-FASN antibody (dilution 1:100, cat. no. 10624-2-AP, ProteinTech Group, Inc.). Peroxidase-labeled secondary antibodies were applied, and diaminobenzidine (DAB; Zhongshan) staining was performed at 4°C for 10 sec. The slides were then counterstained with hematoxylin at room temperature for 4 min and mounted. As a negative control, samples were analyzed without the primary antibody.
Western blot analysis
To determine the FASN level, proteins were extracted from the tissues by suspension in radioimmunoprecipitation assay (RIPA) buffer (Solarbio). The samples were centrifuged at 13,523 × g at 4°C for 38 min, and the supernatants were recovered for analysis. The protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Sigma-Aldrich; Merck KGaA). Protein samples (40 µg) were electrophoresed on a pre-cast bis-Tris polyacryl-amide gel (8-12%) and then transferred onto a polyvinylidene difluoride membrane (EMD Millipore). The membranes were blotted with rabbit anti-FASN (1:1,000) and mouse anti-actin (all from ProteinTech Group, Inc.) followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000; ZsBio). Immunoblots were visualized using enhanced chemiluminescence (LAS-4000; GE Healthcare).
mRNA expression analysis
The expression of the FASN gene was analyzed by RT-qPCR. RNA was isolated from 40 frozen samples (20 from Wilms’ tumors and 20 from adjacent tissues) from which sufficient material was obtained with the miRVana miRNA Isolation Kit (TaKaRa Bio, Inc.). The quantity and quality of total RNA was determined with a Nanodrop ND-2000 Spectrophotometer (Thermo Fisher Scientific, Inc.). RT (37°C 15 min, 85°C 5 sec, and held at 4°C until use) was performed using the TaqMan Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) in the GeneAmp PCR 9700 system and RT-qPCR amplification with the TaqMan Universal PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). All RT-qPCR measurements were obtained in a 7900HT Fast Real Time PCR System with the ExpressionSuite Software v1.0 (Applied Biosystems; Thermo Fisher Scientific, Inc.). All siRNAs were purchased from Personalbio, and their sequences were as follows: 5′-CTC ATCAAGTGGGACCACAG-3′ (forward) and 5′-CAGCGT CTTCCACACTATGC-3′ (reverse) for FASN; 5′-AATCGT GCGTGACATTAAGG-3′ (forward) and 5′-TAGTTTCGTGGATGCCACAG-3′ (reverse) for actin. RT-qPCR analysis was performed using SYBR Premix Ex Taq™ II (Takara, Japan), according to the manufacturer′s protocol. The qPCR reactions were performed on a Roche 480 Real-Time PCR System using the standard procedure for two-step PCR amplification as follows: Pre-denaturation at 95°C for 30 sec, PCR at 95°C for 5 sec, 60°C for 30 sec, for 40 cycles; melting curve: 95°C for 5 sec, 60°C for 60 sec, 95°C for 15 sec; cooling: 50°C for 30 sec. After the reaction, the amplification curve and melting curve of qPCR were confirmed and data were analyzed (18). The FASN mRNA expression levels were examined using the 2-ΔΔCq method and were compared against the level of actin (19).
Immunofluorescence
Cell concentration smears were performed and the cells were fixed with 4% paraformal-dehyde at room temperature for 20 min. The cells were thoroughly rinsed with 0.01 M PBS (5 min × 3) and incubated in 0.3% Triton at room temperature for 20 min, followed by washing in 0.01 M PBS (5 min × 3). Next, 30 µl/sample goat serum blocking solution was added at room temperature for 60 min followed by primary antibody at 30 µl/sample (1:100 diluted in goat serum). The cells were placed in a wet box at 4°C overnight and then washed in 0.01 M PBS (5 min × 3). On the next day, fluorescent secondary antibody (dilution 1:100) was added at 30 µl/sample (10% skimmed milk powder) at room temperature in the dark for 60 min, followed by addition of 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. Next, the cells were washed in 0.01 M PBS (5 min × 3) and mounted with an anti-fluorescence quencher.
Statistical analysis
The data were statistically analyzed using Student’s t-test, the χ2 test, or Fisher’s exact test using SPSS version 19.0 (SPSS, Inc.). The survival curves were analyzed by the Kaplan-Meier method. P<0.05 was considered to indicate statistically significant differences. Tandem mass spectra were processed by PEAKS Studio version 8.0 (Bioinformatics Solutions, Inc.). Differentially expressed proteins were filtered if their fold change was >2 and their P-value was <0.01 (significant >20). The P-value for IPA data analysis was based on the Right-Tailed Fisher’s Exact Test algorithm.
Results
MS revealed 19 differentially expressed lipid metabolism-related enzymes in tumors and adjacent tissues
Label-free proteomics analysis revealed that there were 219 upregulated and 218 downregulated proteins in the tumor (Fig. 1). The HCA map indicates the extent of the peptide or protein expression in terms of depth of color, and the GO classification results can be visualized using bar graphs. The GO classification bar graph uses the top 20 pathways with the smallest P-value (Fig. 1). The results were divided into biological processes, molecular functions and cellular components. As shown in Fig. 2, 437 proteins were input into STRING software, and there were associations among 418 of those proteins. Subsequently, the DAVID functional annotation tool was used to perform protein functional analysis. Among the 437 proteins, 19 were associated with lipid metabolism, including lipid synthesis and decomposition (Fig. 3B). The upregulated proteins included FASN, inositol-3-phosphate synthase 1 (ISYNA1), and arachi-donate-15-lipoxygenase (ALOX15), and the downregulated proteins included hydroxyacyl coenzyme A dehydrogenase beta [HADHB (Fig. 3C)], acetyl coenzyme A acyltransferase 1 (ACAA1), acetyl coenzyme A acyltransferase 2 (ACAA2), carnitine palmitoyltransferase 1A (CPT1A), enoylcoen-zyme A hydratase (ECHS1), acyl-CoA synthetase family member 2 (ACSF2), enoyl-coenzyme A, hydratase/3-hydroxy-acyl coenzyme A dehydrogenase (EHHADH), pyruvate carboxylase (PC), acyl-CoA dehydrogenase medium-chain (ACADM), short/branched chain acyl-CoA dehydrogenase (ACADSB), enoyl coenzyme A hydratase 1 (ECH1), GM2 ganglioside activator (GM2A), 2,4-dienoyl-CoA reductase 1 (DECR1), hydroxyacyl coenzyme A dehydrogenase alpha [HADHA (Fig. 3C)], ubiquinone oxidoreductase subunit AB1 (NDUFAB1) and histone H3.3 [H3F3A (Fig. 4)].
KEGG pathway analyses for lipid metabolism
Fatty acid, triacylglycerol and ketone body metabolism pathway analysis uncovered lipid metabolism-related proteins or protein complexes, small molecules, and their interactions (Fig. 3). KEGG enrichment provides plotting of the top 20 pathways with the smallest P-value. The ordinate indicates the percentage of total protein in the pathway. Fatty acid metabolism and fatty acid degradation are included among the top 20 most highly expressed signaling pathways. The KEGG signaling pathway demonstrated the pathways of fatty acid elongation and fatty acid degradation (the EC code is used in the figure instead of the related enzyme). It was observed that the HADH, HAHDB, ACAA1 and ACAA2 proteins all hold important positions in the KEGG signaling pathway and participate in this process.
Quantitative informatics analysis of IPA
IPA analysis revealed that protein metabolism in Wilms’ tumors is closely associated with tumor cell death, cell cycle, and lipid metabolism (Fig. 5). Lipid metabolism plays an important role in tumors, and signaling pathway analysis has demonstrated that dysregulated proteins play an important role in fatty acid metabolism and are involved in the classical pathways, apoptosis and autophagy of multiple tumors (Fig. 5). Signaling pathway analysis revealed that dysregulated proteins play an important role in fatty acid metabolism pathways, and that sirtuin plays a key role in multiple tumors (Fig. 6). It may be inferred from the regulatory sub-effector network that FASN is involved in cell migration, cell proliferation and tumor necrosis at important positions in the total regulatory network and is implicated in various signaling pathways (Fig. 7).
Validation of FASN expression between Wilms’ tumor and adjacent tissues
Next, MS was used to determine whether the expression of FASN in tumors was higher compared with that in adjacent tissues (Fig. 2). Analysis of the Oncomine database, RT-qPCR, immunohistochemistry, western blotting and immunofluorescence were further performed to verify the results (Figs. 8 and 9). Oncomine database analysis demonstrated that FASN gene expression was significantly increased in Wilms’ tumors (P=0.005 vs. normal tissue; Fig. 8A). Then, RT-PCR was used to detect the expression of FASN in 20 pairs of tumors and adjacent tissues, and the expression of FASN was found to be upregulated in Wilms’ tumors (P<0.01; Fig. 8B). The results of immunohistochemistry, western blotting and immunofluorescence all verified that the expression of the FASN protein was significantly increased in tumors compared with that in adjacent tissues.
FASN expression and its correlation with clinicopathological parameters
The protein expression of FASN and the clinicopathological parameters of the patients are summarized in Table I. Among Wilms’ tumor tissues, positive staining signals of FASN were detected in 34 samples (52.31%) with high immunoreactivity and 31 (47.69%) with low immunoreactivity. The association between FASN expression and tumor stage (III vs. I, P= 0.001; II vs. I, P=0.007) and histopathological type (P=0.032) was statistically significant. There was no significant correlation between FASN expression and patient sex or age (P=0.620 and P=0.083, respectively).
Survival analysis
Postoperative follow-up was performed for the 65 Wilms’ tumor patients (Fig. 10). In the univariate analysis, the Kaplan-Meier survival curves demonstrated that the survival of patients with high FASN expression was significantly shorter compared with that of patients with low FASN expression (P=0.016).
Discussion
Wilms’ tumor is the most common pediatric malignant tumor of the urinary system, and its incidence rate is ~1/10,000 (20). As the tumor site is concealed, the tumor is usually diagnosed at an advanced stage, and there are currently no specific tumor markers. Wilms’ tumor poses a serious threat to the patients’ wellbeing and the search for novel anticancer targets to improve the clinical efficacy of Wilms’ tumor treatment has been attracting increasing attention.
Lipid metabolism is an important and complex biochemical reaction in the body involving numerous enzymes. This process entails the digestion, absorption, synthesis and decomposition of fat in living organisms. Fat is processed into the substances required by the body to ensure the operation of normal physiological functions (21). Lipids are important substances that serve as energy storage and energy supply, and phospholipids are important structural components of biofilms. Currently, due to the high metabolic properties of tumors, the study of energy metabolism in tumors has been attracting increasing attention from researchers, as is the interrelationship between lipid metabolism and the tumor. Lipid metabolism is closely associated with the proliferation, invasion, migration, radiosensitivity and chemosensitivity of various tumors, such as lobular lung, breast and prostate cancer (6,22,23). As regards the energy metabolism of Wilms’ tumors, current research mainly focuses on glucose metabolism, whereas lipid metabolism has been less extensively studied. In our experiments, preliminary screening was performed by MS of tumor and adjacent tissues. Among the 437 proteins identified, 19 lipid metabolism-related proteins were differentially expressed. When these 19 lipid metabolism proteins were analyzed using KEGG, 11 proteins were found to be involved in the process of long chain elongation and degradation of fatty acids. Certain proteins exhibited synergy, such as EHHADH and HAHDA, which are involved in the conversion of (S)-3-hydroxy-dodecanoyl-CoA to 3-oxo-decanoyl-CoA. The 19 differentially expressed proteins were further analyzed: A total of 16 proteins exhibited low expression (HADHB, ACAA1, ACAA2, CPT1A, ECHS1, ACSF2, EHHADH, PC, ACADM, ACADSB, ECH1, GM2A, DECR1, HADHA, NDUFAB1 and H3F3A), and 3 proteins (FASN, ISYNA1 and ALOX15) were highly expressed. In addition, these 16 proteins and ALOX15 are associated with the metabolism of lipids. FASN and ISYNA1 are involved in the process of lipid synthesis. To a certain extent, lipid synthesis is active and degradation is inhibited in Wilms’ tumors. The reason for this finding may be that the majority of the tissues we selected were from children who had not undergone chemoradiotherapy and the tumor was not under hypoxic conditions. Excessive glucose metabolism (24) may inhibit lipid degradation to a certain extent. However, phospholipids are the main constituents of cell membranes, and the proliferation of tumors is a highly active process that requires the support of lipid synthesis, which may explain the overexpression of synthetase.
IPA is an integrated biological pathway analysis software based on cloud computing. IPA analysis includes function, disease correlation analysis, biological downstream effect analysis, classical pathway correlation analysis, and pathway activity effect prediction (25,26). Through IPA analysis, the dysregulated proteins screened by MS were found to be mainly involved in biological processes, such as cell death, cell cycle progression and lipid metabolism. Although the level of lipid metabolism is relatively lower in tumors compared with that in the adjacent tissues, lipid metabolism plays an important role in tumors; signaling pathway analysis demonstrated that dysregulated proteins play an important role in fatty acid metabolic pathways in multiple tumors. The classical pathway is involved in tumor apoptosis and autophagy, and the dysregulated protein is implicated in the process of post-translational modification. Sirtuin (NAD-dependent deacetylase) plays a key role in the epigenetic regulation of gene expression by altering chromatin structure. This protein has been reported to deacetylate histones and non-histones. It has been demonstrated that sirtuin deacetylation of histones induces chromatin condensation, whereas acetylation of histone acetyltransferase leads to chromatin decondensation. This balance is crucial for maintaining normal cellular function, and any disruption in this process may lead to tumorigenesis (27). Sirtuin-mediated protein deacetylation has been considered to play an important role in cancer (28), and there are reports of dylinin expression disorders in a variety of human malignancies (29-31). IPA results revealed that sirtuin may be dysregulated in Wilms’ tumors, and the fatty acid metabolism pathway may affect biological behaviors, such as proliferation, apoptosis and autophagy of Wilms’ tumors by sirtuin. In addition, the dysregulated proteins can be identified from the regulatory sub-effector network: FASN, one of the lipid synthesis-related enzymes among the dysregulated proteins, holds an important position in the total regulatory network and is involved in processes such as cell migration, cell proliferation and tumor necrosis. In addition, FASN is associated with a variety of signaling pathways.
Several other metabolic proteins in Wilms’ tumors have been found to be reduced compared with their levels in normal tissues, such as aldehyde dehydrogenase and proteins associated with propionic acid and butyrate metabolism and long-chain fatty acid metabolism (24). FASN is a multi-enzyme protein that catalyzes fatty acid synthesis. This protein is not a single enzyme but a whole enzymatic system composed of two identical 272-kDa multifunctional polypeptides (32,33). Accumulating evidence shows that FASN expression is upregulated in various human cancers and confers a survival advantage to these cancer cells (12,34-36). FASN overexpression has also been associated with resistance to anticancer treatments, and FASN has been found to be closely associated with the prognosis of a variety of tumors (37,38); however, its role in Wilms’ tumors has not been extensively investigated. The present study revealed high expression of FASN by MS screening. This finding was further verified by Oncomine database, RT-qPCR, western blot, immunofluorescence and immunohistochemistry analyses, which confirmed the high expression of FASN in Wilms’ tumors. Furthermore, the clinical case parameters of 65 children with Wilms’ tumors were analyzed and it was observed that the expression of FASN was closely associated with tumor stage and pathological type, which confirmed that FASN may be implicated in the malignant progression of Wilms’ tumors. In addition, the results of the survival analysis revealed that the prognosis of patients with high expression of FASN was relatively poor, which may be used as a biomarker to predict the prognosis. Therefore, the findings of the present study may provide a theoretical basis for subsequent experiments in vivo and in vitro, and help identify new targets for the treatment of Wilms’ tumors.
Reprogramming cellular energy metabolism is one of the major markers of cancer, and cancer cells rely on fatty acids as a building block for cell proliferation; thus, disrupting key enzymes or modulators in cellular metabolism may be a promising new approach to cancer therapy (39,40). Targeting lipid metabolism has provided novel ideas for the treatment of tumors (24). In particular, there is an increasing number of studies on targeted therapies for FASN, and inhibition of FASN expression may be of therapeutic value in a variety of tumors. In addition to the specific inhibitors of FASN, such as C75, Fasnall, and EGCG (41-43), which may exert antitumor effects, researchers committed to design specific drugs for FASN and discovered a new antitumor drug, 'compound 34’ for treating various tumors (44). Other studies have also sought to treat tumors by identifying upstream regulators of FASN or miRNAs (7,45). In other tumors, the study of FASN is not limited to clinical specimens; through cell and animal experiments, its mechanism of action has been investigated and was found to include the AKT/mTOR signaling pathway, which is closely associated with tumors (46,47). To a certain extent, these previous studies may explain the effectiveness of targeting FASN for tumor treatment. In addition, the present study may also be of value as a guide for the treatment of Wilms’ tumors, for which the targeted inhibition of FASN may prove useful.
Although extensive studies on cancer and its initiation are beginning to unravel the role of lipid metabolism in this process, the lipid metabolism-related enzymes that are implicated in Wilms’ tumor have not been determined. Due to the low incidence of pediatric vs. adult tumors, there were relatively few cases included in the present study. However, the abovementioned MS screening may provide novel ideas for the study of lipid metabolism in Wilms’ tumors, which may serve as a reference for subsequent research. In addition, FASN appeared to be a useful biomarker for Wilms’ tumors based on the analysis of the prognosis of 65 pediatric patients. This index provides theoretical support for the prognosis assessment of children with Wilms’ tumors. Moreover, FASN may prove to be useful in the targeted therapy of Wilms’ tumor.
Funding
The present study was supported by funding provided by the National Natural Science Foundation (grant no. 81400575), the Hunan Provincial Natural Science Foundation of China (grant no. 2017JJ4071), the Science and Technology Development Plan Project of Shandong Province, China (grant nos. 2014GSF118144, 2018GSF118209 and 2019GSF108061), Jinan Science and Technology Bureau (grant no. 201602170) and the Shandong Provincial Natural Science Foundation (grant nos. ZR2017MH091 and ZR2015HM048).
Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
Authors’ contributions
XQW and GQD conceived and designed the study. XQW, YDW and YFZ contributed to the work by designing and performing the experiments, collecting and analyzing data, and drafting the manuscript. FG collected patient samples and conceived the experiments. WL and RDW read and approved the final manuscript.
Ethics approval and consent to participate
The study was approved by the Ethics Committee of the Provincial Hospital affiliated to Shandong University (approval no. 2019-124). All the included specimens were obtained following informed consent by the patients’ guardians, and the patient data were anonymized. The research protocol conformed to the principles outlined in the Declaration of Helsinki.
Patient consent for publication
Not applicable.
Competing interests
All the authors declare that they have no competing interests.
Acknowledgments
Not applicable.
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