Contributed equally
Wilms’ tumor is one of the most common malignant tumors of the abdomen in children. However, there is currently no recognized specific biomarker for the clinical diagnosis and prognosis of this tumor. Lipid metabolism is involved in membrane synthesis and oxidation in tumor cells. This process plays an important role in the development of tumors, but it has not yet been investigated in Wilms’ tumor. The aim of the present study was to characterize the changes in lipid metabolism and to contribute to the diagnosis and prognosis of Wilms’ tumor. Proteomics analysis was performed to detect lipid-metabolizing enzymes in 9 tissue samples from Wilms’ tumors and adjacent tissues, and proteomics revealed the presence of 19 differentially expressed lipid-metabolizing enzymes. Protein interaction analysis with the Search Tool for the Retrieval of Interacting Genes/Proteins was used to identify the interacting proteins. Immunohistochemistry (IHC), immunofluorescence and western blotting were used to further confirm whether the expression of fatty acid synthase (FASN) was significantly increased in the tumor tissues. Oncomine database and reverse transcription-PCR analyses further confirmed that the expression of FASN at the gene level was significantly increased in the tumors. Following collection of 65 pediatric cases of Wilms’ tumor at the Shandong Provincial Hospital between 2007 and 2012, the association between the expression of FASN and the clinical characteristics was analyzed, and IHC analysis further demonstrated that FASN expression was significantly associated with tumor stage and size. The association between FASN and the prognosis of children with Wilms’ tumor was analyzed using Kaplan-Meier survival curves. In addition, univariate survival analysis revealed that higher expression of FASN in Wilms’ tumors was associated with poorer prognosis. Our findings revealed that FASN may be used as a prognostic biomarker in patients with Wilms’ tumor. Furthermore, lipid metabolism may play an important role in the occurrence and development of Wilms’ tumor.
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 (
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 (
Fatty acid synthase (FASN) is essential for
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.
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
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
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
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.
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 (
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 (
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.
The protein-protein interaction network was analyzed using STRING v.10 (
The differentially expressed proteins were also identified using DAVID functional annotation for functional analysis (
Pathway analysis was processed by KOBAS (
IPA is a common tool used to model, analyze and understand complex biological systems, particularly to reveal different signaling networks and biological processes (
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-
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
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 (
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
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.
Label-free proteomics analysis revealed that there were 219 upregulated and 218 downregulated proteins in the tumor (
Fatty acid, triacylglycerol and ketone body metabolism pathway analysis uncovered lipid metabolism-related proteins or protein complexes, small molecules, and their interactions (
IPA analysis revealed that protein metabolism in Wilms’ tumors is closely associated with tumor cell death, cell cycle, and lipid metabolism (
Next, MS was used to determine whether the expression of FASN in tumors was higher compared with that in adjacent tissues (
The protein expression of FASN and the clinicopathological parameters of the patients are summarized in
Postoperative follow-up was performed for the 65 Wilms’ tumor patients (
Wilms’ tumor is the most common pediatric malignant tumor of the urinary system, and its incidence rate is ~1/10,000 (
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 (
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 (
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 (
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 (
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.
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).
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
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.
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.
Not applicable.
All the authors declare that they have no competing interests.
Not applicable.
Label-free quantification and analysis. (A) The volcano plot is a scatter plot with the log2 value of the fold change as the horizontal axis and the −log10 change of the P-value as the vertical axis. The data are mainly divided into three major points according to the threshold of the significant change. Red dot, upregulation (219 proteins); blue dot, downregulation (218 proteins); and green dot, unchanged (Wilms’ tumor vs. adjacent tissues). (B) The heat map, also referred to as hierarchical cluster analysis, indicates the extent of the peptide or protein expression in terms of the depth of color. In the vertical axis the sample is clustered, and the peptide is aligned in the horizontal axis. Segment or protein for cluster analysis. (C) Gene Ontology (GO) classification results are visual-ized using bar graphs. The GO classification bar graph uses the top 20 pathways with the smallest P-value. The ordinate indicates the number of proteins in each secondary classification and their percentage of the total number of differential proteins. The color indicates different primary classifications. Biological process (green), molecular function (red); and cellular component (blue). As marked in the figure, Group 1 indicates tumor tissue and Group 2 adjacent tissue.
Mass spectrometry shows the interactions of the 437 proteins and the expression level of FASN. (A) Visualization of protein-protein interactions among the dysregulated proteins in Wilms’ tumors using STRING analysis (confidence mode). There was an association among 418 proteins. A total of 437 proteins were input into the STRING software. The lines represent interactions between proteins. (B) Mass spectrometry demonstrated the expression of FASN. FASN expression was found to be upregulated in tumor tissues (**P<0.01 vs. adjacent tissue). Data are expressed as the mean ± standard deviation of 3 independent experiments. FASN, fatty acid synthase; STRING, Search Tool for the Retrieval of Interacting Genes/Proteins.
KEGG analysis of the 437 differentially expressed proteins. (A) The KEGG enrichment results can be visualized by bar graphs, and the top 20 pathways with the smallest P-value are plotted. The ordinate indicates the percentage of total protein in the pathway. The darker the color, the smaller the P-value. ***P<0.001. (B) The selected enzymes are involved in the process of long-chain fatty acid extension. (C) The selected enzymes are involved in the process of fatty acid degradation. The EC code is concluded, such as ACAA [EC:2.3.1.16], ECHS1 [EC:4.2.1.17], ACSF2 [EC:6.2.1.2], EHHADH [EC:4.2.1.17&1.1.1.35&5.3.3.8], ACADM [EC:1.3.8.7], ACADSB [EC:1.3.99.12], ECH1 [EC:5.3.3.21], DECR1 [EC:1.3.1.34], HADHA [EC:4.2.1.17&1.1.1.211] (
STRING analysis shows the interactions of the 19 lipid metabolism-related proteins. (A) Visualization of protein-protein interactions of the proteins in Wilms’ tumors using STRING analysis (confidence mode) revealed the associations among the 19 proteins. Lines represent interactions between proteins. (B) Statistical data of the expression of the 19 lipid metabolism-related protein. (C) Western blotting of hydroxyacyl coenzyme A dehydrogenase (HADH)A and HADHB revealed marked interaction with fatty acid synthase expression in Wilms’ tumor and its adjacent tissue (*P<0.05 and **P<0.01). Actin was used as a loading control.
IPA of 437 dysregulated proteins in Wilms’ tumor and adjacent normal tissues. IPA can replace the z-score of each major class and its subclasses with orange and blue, and the P-value is replaced by the block size for clustering. IPA, Ingenuity Pathway Analysis.
Post-translational modification involved in the regulation of dysregulated proteins by IPA. IPA scientists have manually mapped >800 biological signaling pathways and metabolic pathways, scoring the most relevant dysregulation pathways with experimental data in canonical pathways analysis. The Overlay and Path Designer tools personalize the editing and extension of the path. In addition, IPA’s unique z-score algorithm also covers the analysis of activation inhibition regulation of nearly 200 pathways to quickly identify relevant pathways with obvious imbalances. Sirtuin (NAD-dependent deacetylase) is involved in the process of protein deacetylation, which includes important processes, such as fatty acid metabolism. IPA, Ingenuity Pathway Analysis.
IPA further clusters the biological functions that may be exerted by each sub-network of transcriptional regulators to find a common regulatory-function network. The important role of FASN in the overall regulatory network is through its involvement in processes including cell migration, cell proliferation and tumor necrosis, and its association with a variety of signaling pathways. IPA, Ingenuity Pathway Analysis; FASN, fatty acid synthase.
Oncomine database and RT-qPCR analyses were used to verify the expression of FASN at the gene level. (A) According to the Oncomine database, FASN gene expression was significantly increased in Wilms’ tumors (P=0.005 vs. normal tissue). (B) RT-PCR was used to examine FASN expression in 20 pairs of tissues, including 10 Wilms’ tumors and respective adjacent tissues. FASN gene expression was found to be upregulated in tumor tissues (**P<0.01 vs. adjacent tissue). Data are expressed as the mean ± standard deviation of 3 independent experiments. FASN, fatty acid synthase; RT, reverse transcription; qPCR, quantitative polymerase chain reaction.
Molecular biology experiments were performed to verify the protein expression of FASN. (A) Western blotting of FASN expression in Wilms’ tumor and its adjacent tissue. Actin was used as a loading control. (B) Quantification was performed by densitometric analysis and normalized to actin levels. **P<0.01 vs. adjacent tissue. Data are expressed as the mean ± standard deviation of 3 independent experiments. (C) Immunohistochemistry of FASN expression in Wilms’ tumor and adjacent tissues. (D) Immunofluorescence of FASN expression in Wilms’ tumor and adjacent tissues. FASN, fatty acid synthase.
Survival curve analysis of the association between FASN and prognosis. The survival curves were analyzed by the Kaplan-Meier method. The follow-up time was 60 months. High expression of FASN indicated poor prognosis in Wilms’ tumor patients (P=0.016 vs. low expression). FASN, fatty acid synthase.
Correlation of expression levels of the FASN protein with the clinicopathological parameters of patients with Wilms’ tumor.
Clinicopathological parameters | n (%) | FASN expression
|
P-value | |
---|---|---|---|---|
High | Low | |||
Sex | 0.620 | |||
Male | 35 (53.8) | 17 | 18 | |
Female | 30 (46.2) | 17 | 13 | |
Age (months) | 0.083 | |||
>24 | 28 (43.1) | 11 | 17 | |
≤24 | 37 (52.9) | 23 | 14 | |
Stage | ||||
I | 32 (49.2) | 9 | 23 | |
II | 21 (32.3) | 16 | 5 | 0.001 (II vs. I) |
III | 12 (18.5) | 9 | 3 | 0.007 (III vs. I) |
Histopathological type | 0.032 | |||
Favorable | 46 (70.8) | 20 | 26 | |
Unfavorable | 19 (29.2) | 14 | 5 | |
Lymphatic metastasis | 0.086 | |||
Yes | 10 (15.4) | 8 | 2 | |
No | 55 (84.6) | 26 | 29 |
FASN, fatty acid synthase.