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ORIGINAL ARTICLE
Year : 2021  |  Volume : 17  |  Issue : 5  |  Page : 1281-1285

Roles of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha, a lipid metabolism enzyme, in Wilms tumor patients


1 Department of Pediatric Surgery, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China
2 Department of Pediatric Surgery, Zibo Municipal Hospital, Zibo, China

Date of Submission16-Aug-2021
Date of Decision21-Sep-2021
Date of Web Publication24-Nov-2021

Correspondence Address:
Feng Guo
Department of Pediatric Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, 324 Jingwu Street, Jinan 250021, Shandong
China
Wei Liu
Department of Pediatric Surgery, Shandong Provincial Hospital Affiliated to Shandong First Medical University, 324 Jingwu Street, Jinan 250021, Shandong
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.jcrt_1388_21

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 > Abstract 


Objectives: Wilms tumor is a common pediatric malignant tumor that accounts for approximately 95% of kidney tumors in children. The role of lipid metabolism in tumors has attracted increased attention in recent years. We examined the role of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha (HADHA), a lipid metabolism enzyme, in the pathogenesis of Wilms tumor.
Materials and Methods: In a previous study, we screened Wilms tumors and adjacent normal tissues for differentially expressed proteins by mass spectrometry and verified the results by western blot analysis. The Oncomine database and quantitative reverse transcription–polymerase chain reaction were used to verify the expression of HADHA at the genetic level. Immunohistochemistry and immunofluorescence were also used to validate the differential expression of the HADHA protein. The relationship between histopathological typing, clinical pathology, and HADHA expression was analyzed in 65 paraffin-embedded specimens from pediatric Wilms tumor patients. Kaplan–Meier survival curves were used to analyze the relationship between the expression of HADHA and patient prognosis.
Results: HADHA was expressed at low levels in Wilms tumor tissue compared with the corresponding normal tissue. The expression of HADHA was closely associated with histopathological typing (P = 0.030). The prognostic analysis of 65 children with Wilms tumor showed that high expression of HADHA was closely associated with poor prognosis (P = 0.046).
Conclusions: HADHA expression is downregulated in Wilms tumor tissues, but high expression in tumor tissues is associated with clinical stage and the prognosis of children with this tumor.

Keywords: Clinicopathological parameters, hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha, lipid metabolism, prognosis, Wilms tumor


How to cite this article:
Wu X, Feng R, Wang X, Guo F, Liu W. Roles of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha, a lipid metabolism enzyme, in Wilms tumor patients. J Can Res Ther 2021;17:1281-5

How to cite this URL:
Wu X, Feng R, Wang X, Guo F, Liu W. Roles of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha, a lipid metabolism enzyme, in Wilms tumor patients. J Can Res Ther [serial online] 2021 [cited 2022 Jan 17];17:1281-5. Available from: https://www.cancerjournal.net/text.asp?2021/17/5/1281/331103

Xiangyu Wu and Run Feng contributed to the paper equally.





 > Introduction Top


Wilms tumor is one of the most common malignant tumors of the abdomen in children. The peak age of onset is 3–4 years old and 80% of patients are diagnosed before 5 years of age.[1] Multidisciplinary treatment methods are used for the treatment of Wilms tumor, which results in long-term survival rates that exceed 90%. However, the relatively high incidence of treatment-related complications is currently a challenging problem.[2],[3],[4],[5],[6] Moreover, the mortality rate of children with tumor recurrence is higher than that of those without recurrence.[7] Accurate classification of Wilms tumor is an important step in improving prognosis. Therefore, it is important to examine the relevant factors that affect the prognosis of children with Wilms tumor and to improve treatment efficacy.

Hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha (HADHA) is the alpha subunit of the mitochondrial trifunctional protein, which catalyzes the last three steps of the mitochondrial beta-oxidation of long-chain fatty acids. It is responsible for catalyzing long-chain 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase activities.[8] In addition to fatty acid β-oxidation, HADHA is involved in other lipid metabolic processes, such as lipid biosynthesis, ketone formation, and ketone hydrolysis. It has been reported that HADHA is involved in the pathogenesis, prognosis, and chemoradiation resistance of human cancer. The expression of HADHA is downregulated in breast cancer, especially in metastatic and recurrent breast cancer.[9] The expression of HADHA was reduced in hepatocellular carcinoma compared with that of the nontumor group,[10] and HADHA may be a potential predictor of the response of lung cancer to platinum-based chemotherapy.[11] Recent studies also found that HADHA is involved in miRNA processing,[12] autophagy, and apoptosis.[13],[14],[15] However, despite being a key enzyme in lipid metabolism, relatively little is known regarding the various functions of HADHA, and there has been no data indicating a role for HADHA in Wilms tumor. Whether lipid metabolism, in particular HADHA, is involved in the development of Wilms tumor is also unclear. Therefore, we explored the role and regulatory mechanism of key lipid metabolism enzymes in Wilms tumor to further elucidate its underlying pathogenesis and provide new targets and strategies for treatment.


 > Materials and Methods Top


Tissues

This study was approved by the ethics committees of Shandong Provincial Hospital Affiliated to Shandong First Medical University. A total of 65 patients with Wilms tumor who did not undergo radiotherapy or chemotherapy before radical or palliative nephrectomy from January 2007 to January 2012 were selected for immunohistochemical analysis. Clinical data were recorded including gender, age, tumor size, stage, histopathological type, metastasis, and follow-up information. The patients consisted of 35 males and 30 females with a mean age at diagnosis of 3.2 years (ranging from 0.25 to 11.8 years). A detailed ratio is presented in [Table 1]. The duration of follow-up was 60 months. In addition, 20 cases of Wilms tumor and adjacent tissues were collected from March 2015 to December 2017 and stored at − 80°C. The tumors were graded according to the Fuhrman nuclear grade (G1: 10 cases, G2: 7 cases, G3: 3 cases). Radiotherapy, chemotherapy, and immunotherapy were not administered before surgery and the samples were verified by two pathologists after surgery.
Table 1: Correlation of expression levels of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha protein with clinicopathological parameters of Wilms tumor patients

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The process of genetic screening

As described in our previous article,[16] a total of nine cases (G1, n = 3; G2, n = 3; and G3, n = 3) were subjected to label-free quantification. Using mass spectrometry, we identified 437 differentially expressed proteins between tumor and adjacent tissue. Through GO enrichment and KEGG analysis, we identified several differentially expressed proteins that are involved in lipid metabolism. We then performed a DAVID protein function analysis and found 19 differentially expressed lipid metabolism proteins, which included FASN, ISYNA1, ALOX15, HADHB, ACAA1, ACAA2, CPT1A, ECHS1, ACSF2, EHHADH, PC, ACADM, ACADSB, ECH1, GM2A, DECR1, HADHA, NDUFAB1, and H3F3A. After subsequent verification, we selected HADHA as a candidate for further study.

Immunohistochemistry analysis

Tissues from Wilms tumor samples were subjected to fixation, dehydration, and embedding. The tissues were then cut into sections. After dewaxing and rehydration of the sections, heat-induced epitope retrieval was performed with 1 mM Ethylenediaminetetraacetic acid (pH 9.0) in a microwave oven. Samples were then immersed in a 3% H2O2 solution for 30 min to inhibit endogenous peroxidase. After blocking with goat serum for 30 min, the sections were incubated overnight at 4°C with anti-HADHA antibody (dilution 1:100, 10758-1-AP, Proteintech, USA). Peroxidase-labeled secondary antibodies were applied and diaminobenzidine (DAB; Zhongshan, China) was used for color development. Tissue sections were then counterstained with hematoxylin and mounted on slides. Samples were analyzed without the primary antibody as a negative control.

mRNA expression analysis

The expression of HADHA genes was analyzed by quantitative reverse transcription–polymerase chain reaction (qRT-PCR). RNA was isolated from 40 frozen samples (20 from Wilms tumors and 20 from their adjacent tissues) of which sufficient material was obtained with the miRVana miRNA Isolation Kit (TaKaRa Bio, Inc., Japan). The quantity and quality of the total RNA were determined with a Nanodrop ND-2000 spectrophotometer (Thermo Scientific). Reverse transcription was performed using the TaqMan Reverse Transcription Kit (Applied Biosystems; Life Technologies) and the GeneAmp PCR 9700 system, and qRT-PCR was performed with TaqMan Universal PCR Master Mix (Applied Biosystems). All qRT-PCR measurements were obtained using a 7900HT fast real-time PCR System with Expression Suite Software v1.0 (Applied Biosystems). All siRNAs were purchased from Personalbio and the sequences were as follows: 5'-GAGCTCCACAGAAGGATGTT-3' (forward) and 5'-CAGTCAAGTTGCTGAAGATG-3' (reverse) for HADHA 5'-AATCGTGCGTGACATTAAGG-3' (forward) and 5'-TAGTTTCGTGGATGCCACAG-3' (reverse) for β-actin.

Immunofluorescence

Following dewaxing and rehydration, heat-induced epitope retrieval was performed with 1 mM Ethylenediaminetetraacetic acid (pH 9.0) in a microwave oven. Slides were incubated in 0.3% Triton X-100 at room temperature for 20 min, followed by a wash in 10 mM PBS (5 min × 3). Next, 30 μL of goat serum blocking solution was added at room temperature for 60 min, followed by a 1:100 dilution of primary antibody at 30 μL per sample (diluted in goat serum). The cells were placed in a wet box at 4°C overnight and then washed with 10 mM PBS (5 min × 3). The following day, a 1:100 dilution of fluorescent secondary antibody was added at 30 μL per sample (10% skim milk powder) and incubated at room temperature in the dark for 60 min, followed by 4',6-diamidino-2-phenylindole staining, which was applied for 10 min. Next, the cells were washed with 10 mM PBS (5 min × 3) and mounted with an anti-fluorescence quencher.

Statistical analysis

The data were analyzed using a Student's t-test, Chi-square test, or Fisher's exact test with SPSS version 19.0 software (IBM SPSS, NY, USA). The survival curves were analyzed by the Kaplan–Meier method. P < 0.05 was considered statistically significant.


 > Results Top


The results of label-free mass spectrometry from our previous study

The results of label-free mass spectrometry from our previous report showed that compared with the corresponding normal tissues adjacent to the tumor,[16] the expression of HADHA in Wilms tumor tissue was low [Figure 1]. The western blot results verified this finding. Moreover, KEGG pathway analyses also showed that proteins, including HADHA, HADHB, ACAA1, and ACAA2, were all located at important positions in the relevant KEGG signaling pathway and participate in the process.[16]
Figure 1: Mass spectrometry showing the expression of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha in tumor and normal tissues. (a) String database shows the protein-protein interactions of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha. Of these, HADHB, ACAA1, ACAA2, and ACADM, are differentially expressed proteins from our previous report that have a mutual relationship with hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha (https://string-db.org/cgi/network.pl?taskId=8avogdOagemI). (b) Mass spectrometry showing the expression of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha. Hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha expression is downregulated in tumor tissues. (**P < 0.01 vs. adjacent tissue. Data are expressed as the mean ± standard deviation of three independent experiments

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Expression of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha in Wilms tumor and adjacent tissues

Analyses using the Oncomine database, qRT-PCR, immunohistochemistry, and immunofluorescence were done to verify the results. Quantitative RT-PCR was used to measure the expression of HADHA (20 pairs of tumors and adjacent tissues were used), which was downregulated in Wilms tumor (P < 0.01) [Figure 2]. The results of immunohistochemistry and immunofluorescence showed that the expression of HADHA protein was significantly increased in adjacent normal tissues compared with tumors [Figure 3] and [Figure 4].
Figure 2: Oncomine database AND quantitative reverse transcription–polymerase chain reaction verifying the expression of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha. (a) Hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha gene expression is shown in the Oncomine database. (b) Quantitative reverse transcription–polymerase chain reaction was used to measure hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha expression in 20 pairs of tissues including 10 Wilms tumors and the respective adjacent tissues. Hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha expression is downregulated in tumor tissues. (**P < 0.01 vs. adjacent tissue. Data are expressed as the mean ± standard deviation of three independent experiments)

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Figure 3: Immunohistochemistry at the protein level to verify the expression of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha. Immunohistochemistry of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha expression in Wilms tumor and adjacent tissues

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Figure 4: (a) in adjacent normal tissue. (b) in Wilms tumor tissue

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Hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha expression and its correlation with clinicopathological parameters

The protein expression and clinicopathological parameters of the patients are summarized in [Table 1]. In Wilms tumor tissues, positive staining for HADHA was detected in 33 (50.77%) tissues with high immunoreactivity and 32 (49.23%) tissues with low immunoreactivity. There was no significant correlation between HADHA expression and patient gender or age (P = 0.209, P = 0.137). There was no correlation between HADHA expression and tumor stage (Phase II vs. I Stage P = 0.406; Stage III vs. Stage I P = 0.300) or lymph node metastasis (P = 0.479). However, the correlation between HADHA expression and histopathological type (P = 0.030) was statistically significant.

Survival analysis

Postoperative follow-up was carried out for the 65 patients with Wilms tumor [Figure 5]. In a univariate analysis, the Kaplan–Meier survival curves revealed that the survival of patients with high HADHA expression was significantly shorter compared with that of patients with low HADHA expression (P = 0.046).
Figure 5: Survival curve analysis of the relationship between hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha and prognosis. The survival curves were analyzed by the Kaplan–Meier method. The follow-up time was 60 months. High expression of hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha is associated with poor prognosis in patients with Wilms tumor (P = 0.046 vs. low expression patients)

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 > Discussion Top


Wilms tumor is the most common solid malignant tumor of the abdomen in children.[17] Although many prognostic biomarkers have been shown to be effective for predicting the prognosis of tumors in adults,[18] there are relatively few studies regarding tumor prognosis in children, and there are no specific, reliable markers or prognostic indicators for Wilms tumor. This study indicates a role for HADHA in Wilms tumor through an analysis of clinical specimens. We selected HADHA based on our previous analysis of the STRING database, in which we identified 19 differentially expressed proteins by mass spectrometry.[16] Furthermore, we used qRT-PCR, western blot analysis, and immunohistochemistry to evaluate differences in the expression of HADHA in tumors and adjacent tissues. The low expression of HADHA in tumors confirmed the mass spectrometry results. However, through our analysis of the clinicopathological parameters of 65 clinical specimens and patient prognosis, we found that children with Wilms tumor that exhibit high HADHA expression have a worse prognosis compared with those showing low expression.

Current comprehensive treatment methods can significantly improve the 5-year survival rate of children with Wilms tumor,[19] but knowledge is lacking regarding the mechanisms of tumor occurrence and development, the development of multidrug resistance, and the severity of chemotherapeutic drug side effects. Moreover, because of the negligence of parents, tumors are often diagnosed late, thus more than half of the tumors are in at middle and advanced stages when they are discovered.[20] Wilms tumor seriously threatens the life and health of children because of the hidden tumor site, rapid development of the disease, and lack of specific tumor markers.[21] If the characteristics of Wilms tumor were more clearly defined based on postoperative pathology, targeted treatment could be developed to greatly reduce the possibility of recurrence. In contrast, children with low risk for recurrence could be prospectively stratified, and excessive radiotherapy and chemotherapy could be avoided. Hence, to a certain extent, this study provides theoretical support for individualized treatment of children with Wilms tumor.

Here, we found that the expression of HADHA in tumors was lower compared with that in adjacent tissues, but the prognosis of children with high HADHA expression was worse. There are several possible reasons for this finding. First, from a pathological point of view, Wilms tumor may result from abnormal proliferation because of the failure of the metanephric embryo base to differentiate into renal tubules and glomeruli. Bove and McAdams suggested that the nephroblastomatosis complex may transform into Wilms tumor.[22] That is, Wilms tumor may co-differentiate and exist with the kidney parenchyma from the embryonic stage rather than originate from the kidney tissue, which may lead to differences in the expression of some genes. Second, the division of tumor cells is often abnormal and active, and lipids are an essential component of cell membranes. In the resting state, the energy supply of tumor cells often depends on glucose and amino acids. Taking these considerations into account, the expression of HADHA in tumors may be reduced. However, the treatment of Wilms tumor requires chemotherapy after surgery, even for children with Stage I tumors. Radiotherapy and chemotherapy create a state of hypoxia in which the tumor lacks energy. The enhancement of lipid metabolism may provide tumor cells with the necessary energy for survival under hypoxia. Therefore, the high expression of HADHA may be a sign of poor prognosis in children.

Due to the low incidence of pediatric tumors, there are relatively few samples (vs. adults) available for our experiments. This is one limitation of our study. It was impossible to group patients in more detail because of the sample size; in particular, we could not perform follow-up grouping studies based on different treatment regimens.


 > Conclusion Top


To summarize, we analyzed specimens from 65 children with Wilms tumor and revealed that HADHA is a useful prognostic marker for Wilms tumors. This finding provides theoretical support for the further assessment of prognosis in children with Wilms tumor. Moreover, HADHA may be a useful lipid metabolism protein to target for Wilms tumor treatment.

Financial support and sponsorship

This study was funded by the National Natural Science Foundation (81400575), the Hunan Provincial Natural Science Foundation of China (grant no. 2017JJ4071), the Science and Technology Development Plan Project of Shandong Province, China (2014GSF118144, 2018GSF118209, 2019GSF108061), Jinan Science and Technology Bureau (201602170), and the Shandong Provincial Natural Science Foundation (ZR2017MH091, ZR2015HM048, ZR2020QH263).

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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Liu S, Liu X, Wu F, Zhang X, Zhang H, Gao D, et al. HADHA overexpression disrupts lipid metabolism and inhibits tumor growth in clear cell renal cell carcinoma. Exp Cell Res 2019;384:111558.  Back to cited text no. 14
    
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