BENTHAM SCIENCE
Denticleless E3 Ubiquitin Protein Ligase Homolog as a Potential Biomarker for Adrenocortical Carcinoma Screening
=== Endocrine, Metabolic & Immune Disorders
Drug Targets
==
Xin Yan1,2,”, Li-Xing Pang1,”, Xiao Lu1, Sheng Chen1, Li Li2, Xing-Huan Liang2, De-Cheng Lu2,* and Zuo-Jie Luo2*
‘Department of Endocrinology, The Second People’s Hospital of Nanning City, The Third Affiliated Hospital of Guangxi Medical University, Nanning 530031, Guangxi Province, China; 2Department of Endocrinology, The First Af- filiated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Province, China
Abstract: Objective: This study aims to investigate the potential of denticleless E3 ubiquitin pro- tein ligase homolog (DTL) as a biomarker for adrenocortical carcinoma (ACC) detection through bioinformatics analysis and experimental validation.
Methods: Differentially expressed genes (DEGs) between ACC and adrenocortical adenoma (A- CA) were identified through bioinformatics analysis. A protein-protein interaction (PPI) network was constructed using Cytoscape software, and core genes were screened with the CytoHubba MCODE plug-in. Survival analysis was performed using the University of ALabama at Birming- ham CANcer (UALCAN) data analysis portal. Immunohistochemistry was employed to assess DTL expression in adjacent normal tissues, ACA, and ACC.
ARTICLE HISTORY
Received: October 25, 2024 Revised: February 19, 2025 Accepted: February 26, 2025
DOI:
10.2174/0118715303334496250518033852
Results: Two gene expression series (GSEs) retrieved from the Gene Expression Omnibus (GEO) database yielded 115 DEGs. Using the PPI network, three core genes were identified, among which DTL and TPX2 were highly expressed in ACC. Notably, DTL had the highest core gene score. Elevated DTL expression in individuals with ACC was significantly associated with a poor prognosis (P < 0.0001). Immunohistochemistry analysis revealed a significantly higher positive expression rate and a strong positive expression rate of DTL in ACC compared to ACA (x2 = 11.708, P < 0.01). The positive expression rate of DTL in both ACC and ACA was significantly higher than in the adjacent normal adrenal cortex (P < 0.01). The expression of DTL followed a gradient, being highest in ACC, followed by ACA, and lowest in the normal adrenal cortex adja- cent to the tumor. Additionally, DTL protein expression was significantly correlated with tumor size and infiltration metastasis (P < 0.05). Individuals with high DTL expression had significantly shorter survival times than those with low DTL expression (P < 0.05).
Conclusion: DTL exhibits potential as a novel biomarker for distinguishing between benign and malignant adrenocortical tumors and may serve as a prognostic indicator for ACC.
Keywords: Adrenocortical carcinoma, bioinformatics, biomarkers, DTL.
1. INTRODUCTION
Adrenocortical carcinoma (ACC) is a rare but aggressive malignancy associated with significant morbidity in patients with adrenocortical tumors, with an estimated incidence of approximately 0.7-2 per million individuals annually [1]. Advances in imaging modalities, such as cross-sectional
imaging techniques, have facilitated the increased detection of adrenal masses during evaluations for non-adrenal condi- tions, commonly referred to as adrenal incidentalomas [2]. Although the majority of adrenal incidentalomas are benign and non-functional, they pose a risk of malignant transforma- tion and endocrine dysfunction. ACC accounts for approxi- mately 15% of adrenal incidentalomas [3].
At present, there is no universally accepted standard for the differential diagnosis of adrenal incidentalomas. The Weiss system remains the most commonly used histopatho- logical gold standard for evaluating ACT malignancy [4].
* Address correspondence to these authors at the Department of Endocrinol- ogy, The First Affiliated Hospital of Guangxi Medical University, No. 6 Shuang Yong Road, Qingxiu District, Nanning, 530021, Guangxi Province, China; E-mails: zuojieluolzj@126.com, ludechengldc@126.com
“These authors contributed equally to this study.
However, its limitations-including interobserver variabili- ty, reduced sensitivity in pediatric populations, and diagnos- tic uncertainty in tumors with intermediate Weiss scores (e.g., scores of 3-4)-are well-documented [5]. A multicen- ter study by Riedmeier et al. revealed that a considerable pro- portion of pediatric ACC cases were misclassified using the Weiss criteria, leading to delayed clinical interventions [4]. These challenges have driven efforts to identify molecular markers that can enhance diagnostic accuracy. Recent ge- nomic profiling studies have identified recurrent mutations in genes such as TP53, CTNNB1, and ZNRF3 in ACC; how- ever, their clinical utility as independent, standalone markers remains constrained by heterogeneity and the lack of stan- dardized assays.
In this context, the exploration of cell cycle regulators as biomarkers offers a promising avenue for improving ACC di- agnosis. Denticleless E3 ubiquitin protein ligase homolog (DTL), also known as CDT2 or DCAF2, is a critical compo- nent of the CRL4Cdt2 ubiquitin ligase complex [6-8]. This complex orchestrates the degradation of key cell cycle regu- lators, including p21, SET8, and Cdt1, thereby ensuring proper DNA replication and damage repair [9]. By ensuring the timely proteolysis of these substrates, DTL maintains ge- nomic stability and prevents replication stress, a mechanism frequently exploited in tumorigenesis [10]. Emerging evi- dence has linked DTL dysregulation to oncogenesis across diverse malignancies. For example, Chen et al. noted that DTL silencing in hepatocellular carcinoma (HCC) induces G2/M phase arrest and suppresses tumor growth through p21 stabilization, highlighting its potential role as a therapeu- tic target [7]. Despite these insights, the functional signifi- cance of DTL in adrenocortical tumors remains unexplored, representing a critical gap in both basic and translational re- search.
To address this knowledge gap, the present study em- ployed an integrative approach combining bioinformatics analysis of public gene expression datasets and immunohisto- chemical validation in clinical specimens. DTL was identi- fied as a key ACC-associated gene through bioinformatics screening, and the expression of DTL in benign and malig- nant adrenocortical tumors was subsequently verified using immunohistochemistry. The relationship between DTL ex- pression and clinicopathological features of adrenocortical tumors, including tumor proliferation activity, was analyzed to explore the role of DTL in the occurrence and develop- ment of human ACC and to provide a theoretical basis for improving the diagnostic accuracy and treatment strategies for ACC.
2. MATERIALS AND METHODS
2.1. Gene Expression Profile Data
Two mRNA expression datasets, GSE12368 and GSE33371, for adult ACC were retrieved and downloaded from the Gene Expression Omnibus (GEO) database (http- s://www.ncbi.nlm.nih.gov/geo/). All mRNA expression pro- files were generated using the GPL570 platform (Affymetrix
Human Genome U113 Plus 2.0 Array). The GSE12368 da- taset consisted of 12 ACC samples and 16 adrenocortical adenoma (ACA) samples, while the GSE33371 dataset in- cluded 33 ACC samples and 22 ACA samples. To ensure da- ta reliability and consistency, background signal correction and normalization preprocessing on the raw CEL files were conducted using the affy package in RStudio (Version 3.5.3). REMARK guidelines were followed.
2.2. Screening of Differentially Expressed Genes (DEGs)
The gene expression matrix was divided into the case group (ACC) and the control group (ACA) for the identifica- tion of differentially expressed genes (DEGs). The statistical significance of gene expression difference was calculated us- ing the limma package in R [11], and the Benjamini-Hoch- berg (BH) method was used for multiple testing correction. DEGs were defined based on the following criteria: P < 0.05, |logFC| ≥ 2. The R software package pheatmap [7] was used to generate a heatmap to visualize the expression pat- terns of the DEGs.
2.3. Functional Annotation and Pathway Enrichment Analysis
To investigate the biological significance of the identi- fied DEGs, the Gene Ontology (GO) [12] and the Kyoto En- cyclopedia of Genes and Genomes (KEGG) [13] pathway en- richment analyses were conducted. Functional enrichment of the upregulated and downregulated DEGs was performed us- ing the Database for Annotation, Visualization and Integrat- ed Discovery (DAVID) [14] (Version 6.8) enrichment analy- sis tool (https://david-d.ncifcrf.gov/). This analysis enabled the identification of key biological processes, molecular functions, cellular components, and signaling pathways asso- ciated with the dysregulated genes in ACC.
2.4. Protein-Protein Interaction (PPI) Network and Mod- ule Selection of DEGs
The Search Tool for Retrieval of Interacting Genes/Pro- teins (STRING) database [15] (Version: 10.0) (http://www.string-db.org/) was utilized to investigate poten- tial interactions among the proteins encoded by the DEGs. The PPI network model of DEGs was constructed using Cy- toscape (Version 3.6.0) [16]. To identify key functional mod- ules in the PPI network, the MCODE plug-in, which detects highly interconnected gene clusters, was used. Additionally, the CytoHubba plug-in was utilized to identify core genes. The top 30 hub genes were selected using four topological ranking methods: radiality, betweenness, stress, and cluster- ing coefficient. The common core genes were identified and ranked using Venn diagram analysis (http://bioinformatics. psb.ugent.be/webtools/Venn/).
2.5. Survival Analysis of the Core Gene DTL
The University of ALabama at Birmingham CANcer (UALCAN) data analysis portal (http://ualcan.path.uab.edu/ analysis.html) was used to evaluate the prognostic signifi- cance of DTL. The survival data were obtained from The
Cancer Genome Atlas (TCGA). Patients with ACC were cat- egorized into high and low DTL expression groups based on the median expression level of DTL (threshold: 3rd quartile). Additionally, UALCAN was used to download the mRNA and clinical data from the TCGA database to evaluate the correlation between DTL expression and tumor grade. This analysis aimed to determine whether DTL could be used as a molecular marker for ACC, particularly about tumor progres- sion and prognosis.
2.6. Expression of DTL in ACC
2.6.1. Clinical Data
A total of 57 paraffin-embedded adrenocortical tumor specimens were obtained from the First Affiliated Hospital of Guangxi Medical University, China, between January 2000 and September 2021. The diagnosis had been con- firmed by pathology after surgical resection and skin adrenal biopsy. Among these, 18 specimens were diagnosed as ACC based on the Weiss histopathological diagnostic criteria [4]. The other 31 cases of ACA and 8 adjacent normal adrenal tissue specimens were included as controls.
The study sample consisted of 49 patients with adreno- cortical tumors (19 males and 30 females) with an average age of 39.5 ± 2.07 years and a male-to-female ratio of 1:1.6. Demographic and Clinicopathological Characteristics of ACC Cases:
The 18 cases of ACC consisted of 3 children under 10 years of age and 15 adults. The tumor diameter was > 5 cm in 16 patients and < 5 cm in 2 patients. As per the TNM stage, there was 1 patient in Stage I, 8 patients in Stage II, 2 patients in Stage III, and 7 patients in Stage IV. Regarding metastatic status, 2 patients had lymph node metastasis, while there were 10 patients without lymph node metastasis and 6 with unclear lymph node metastasis. 3 patients had dis- tant metastasis, and 15 patients without distant metastasis. Seven patients had typical clinical manifestations of hormon- al overproduction, 4 patients showed local compression symptoms, 4 patients had non-specific clinical manifesta- tions, and 3 patients had positive findings detected during routine physical examinations.
The collected pathological specimens were subjected to immunohistochemical (IHC) staining. All histopathological sections were independently reviewed by senior pathologists to ensure diagnostic accuracy.
2.6.2. Determination of Immunohistochemical Results
A double-scoring semi-quantitative integration method based on staining intensity and the proportion of positive cells was used to assess DTL expression in adrenocortical tu- mors. Staining Characteristics
Localization: DTL was primarily expressed in the nu- cleus, with secondary expression observed in the cytoplasm.
Positive Staining: Samples exhibiting clear brown or brown granular deposits were considered positive.
Scoring Criteria:
(A) Staining Intensity Score: The varying degrees of staining of immune-positive cells were scored as follows: no staining was scored as 0 points; mild staining was scored as 1 point; moderate staining was scored as 2 points; and in- tense staining was scored as 3 points.
(B) Percentage of Positive Cells: A total of 10 representa- tive areas were randomly selected from the upper, lower, left, right, and central regions of each slide for cell counting. In each area, the proportion of DTL-positive cells in a total of 1000 cells was scored as follows: < 10% positive cells was considered 0 points; 10%-25% was scored as 1 point; 26%-50% was scored as 2 points; and > 50% was scored as 3 points.
Final Immunohistochemical Score: A semi-quantitative analysis was performed by multiplying the staining intensity (A) by the percentage of positive cells (B). A final score of 0-1 points was considered ”-” (Negative); a score of 2-3 points was considered ”+” (Low expression); a score of 4-6 points was considered ”++” (Moderate expression); and a score of > 6 was considered ”+++” (High expression). For analysis purposes, low expression was indicated by ”-” or ”+” while high expression was indicated by ”++” or ”+++“.
All immunohistochemical slides were independently re- viewed by two senior pathologists in a double-blind manner. Any disagreements were resolved through discussion to ar- rive at a consensus in the final scoring.
2.6.3. Statistical Analysis
All data were digitized and processed using Microsoft Excel for initial collation and SPSS 22.0 for Windows for statistical analysis. The significance level was set at a = 0.05. The chi-square test was used for categorical variable comparisons between groups; Fisher’s exact probability method was applied when expected frequencies in contingen- cy tables were small (n < 5); and the rank sum test (Man- n-Whitney U test or Kruskal-Wallis test) was used for non — parametric comparisons of continuous or ordinal data be- tween groups. GraphPad Prism 7.0 was utilized to perform Kaplan-Meier survival analysis, comparing overall survival (OS) between groups stratified by DTL expression levels. Log-rank tests were used to assess statistical significance in survival differences.
3. RESULTS
3.1. DEGs
A total of 115 DEGs were identified in ACC compared to non-cancerous adrenal cortex tissues. Hierarchical cluster- ing of 45 ACC and 38 ACA samples revealed 62 upregulat- ed and 53 downregulated genes (Figs. 1A and B).
3.2. Cluster Gene Screening
Using Cytoscape, a PPI network consisting of 109 nodes and 772 edges was constructed (Fig. 2A). The most signifi- cant module within this network is shown in Fig. (2B).
WROTYT
B
Volcano
3
2
e
logFC
0
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2.
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0
5
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-log10(adj.P.Val}
A
B
UHRF1
ABLIMI
GSF 11
SEM/GA
RRM2-ZWINT SHCBP1
RM2
ACGAP
CEP55
SLIT2
FABPS
PRC1
FAM83D
CENPK
OLGAP
142830
TPX2
CCNB1
3A32L3
GGH
KIF11
BUB1B
FOXM
KIF4A
PRC1
SLC27/6
CTHRC
COC20
EZH2
TPX2
RMI2
HWR
BUB 1B
COKN3
NR4/2
CCNA2
CENPW UHRF 1RACGAP
CDC20
SHCBP
KIF4A
WELK
NUF2
HSD382
DTL
PBK
UBE20
FLA2G1B
PTTG1
KIFZOA
COK1
CENPV
KIF20A
UBE2T
PTTG1
ANLN
CCNA2
ADS TAP
HOXAS
R
D51AF
EZH2
MAD2L
NDC80
CDK1
ZAD2L
TOPZA
GNS
ZWENT
DTL
UB EXC
KIAA0101
CDKN3
CONB
TTK
NOCCO
ALDH1A1
TVEST
CENPK
MELK
HMMR
NUF2
TOP2A
GINS1
NPYIR
RAV2
UBEZT
KIF11
FOXM1
COH2
ADH18
DLGAP 5
ANLN
CEP55
TTK
KCNQ 1
U0101
C/LB1
PCP4
POK
KCNN2
AOX1
SULF2
TMEM200A
3.3. Core Gene Screening
The four centrality algorithms, namely, radiality, be- tweenness, stress, and clustering coefficient, were applied us- ing the CytoHubba plug-in in Cytoscape to identify the key genes. The top 30 DEGs from each method were extracted, and the four groups of DEGs were intersected using the on- line Venn diagram tool (http://jvenn.toulouse.inra.fr/app/ ex- ample.html). The results identified three core genes: DTL, TPX2, and RAD51AP1 (Fig. 3A). Notably, DTL ranked first across all four algorithms (Fig. 3B), suggesting its pivotal role in ACC pathogenesis.
3.4. Effect of Core Gene DTL Expression on the Progno- sis of Adrenocortical Tumors
Using the UALCAN database, we analyzed the correla- tion between mRNA expression and the overall survival (OS) rate in patients with ACC. High DTL expression was significantly associated with poor prognosis (P < 0.0001) (Fig. 4B). To further validate this, mRNA and clinical data were downloaded from the TCGA database, and UALCAN was used to verify the correlation between DTL expression and tumor grade. The results indicated that DTL expression in Stage IV was significantly higher than that in Stages I, II,
A
B
Betweenness
Stress
Radiality
Betweenness
Stress
ClusteringCoefficient
Radiality
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Clusteringc
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15
3
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20
Size of each bist
25
30
30
30
30
JE
15
30
·
Radality
Betweenness
Stress
ClusteringC
Number of elements: specific (1) or shared by 2, 3, … lists
35
14
18
4 ()
2
TPX2
RAD51AP1
3
1
DTL
A
B
17.5
1.00-
15
Transcript per million
Survival probability
12.5
0.75
10
0.50
7.5
5
2.5
0.25
p<0.001
0
Expression Lavel
- High expression(n=20)
-2.5
0.00
* Low/Medium expression(n=59)
Stage 1 (n=9)
Stage 2 (n=37)
Stage 3 (n=16)
Stage 4 (n=15)
0
1000
2000
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TCGA samples
Time in days
and III (Fig. 4A), suggesting that DTL may play a role in tu- mor progression and metastasis, reinforcing its potential as a prognostic biomarker in ACC.
3.5. Expression and Clinical Significance of DTL in ACC
3.5.1. DTL Expression in Adrenocortical Tumors
The DTL protein was predominantly localized in the nu- cleus of glandular epithelial cells, with brown-positive stain- ing particles. There was a progressive increase in DTL ex- pression from normal adrenal cortex tissue to ACA and ACC (Figs. 5A-C).
In ACC tissues (n = 18), the positive expression rate of DTL was 88.89%, with high expression in 22.22% (4/18 cas- es), low expression in 66.67% (12/18 cases), and decreased or absent in 11.11% (2/18 cases).
In ACA tissues (n = 31), 38.7% (12/31 cases) were posi- tive, but all had low expression, while the remaining 19 cas- es were negative. In normal adrenal cortex tissues adjacent to the tumor, there was almost no positive expression.
The positive and strong positive expression rates of DTL in ACC tissues were significantly higher than in ACA tis- sues (x2= 11.708, P< 0.01) (Table 1). Additionally, the pos- itive expression rate of DTL in ACC was significantly high- er than in adjacent normal adrenal cortex tissues (P < 0.01). DTL positive expression rate in ACA was also significantly higher than in adjacent normal adrenal cortex tissues (P < 0.05) (Table 1).
3.5.2. Correlation Between DTL Expression and Clinico- pathological Features of Adrenocortical Tumors
The expression of DTL protein was not significantly cor-
related with gender, age, or clinical stage (P > 0.05), but there was a significant association with tumor size, invasion, and metastasis (P < 0.05). Patients with a tumor diameter > 5 cm or those with invasion and metastasis showed signifi- cantly higher DTL expression (Table 2).
3.5.3. Correlation Between DTL Expression and Prognosis in ACC
A total of 18 patients with ACC were followed up for 6-89 months (median follow-up time: 39.5 months). Tu- mor-related mortality was 27.8% (5/18 cases). DTL protein expression levels were significantly associated with overall survival (P < 0.05). Patients in the high DTL expression group had a significantly shorter survival time than those in the low DTL expression group. The survival time in the high DTL expression group was (26 ± 7.2) months, while it was (76.3 ± 8.2) months in the low DTL expression group. The 3-year survival rate of patients with ACC also differed signif- icantly, with < 30% in the high DTL expression group and > 80% in the low DTL expression group (Fig. 6). These find- ings suggest that high DTL expression is associated with poorer prognosis in ACC, reinforcing its potential role as a biomarker for tumor progression and prognosis prediction.
4. DISCUSSION
ACC is considered a highly malignant tumor of the en- docrine system, characterized by early metastasis and poor prognosis. Identifying molecular markers of ACC that can be used for early diagnosis and targeted treatment is crucial in order to enhance patient outcomes.
In this study, DTL was highlighted as a key gene impli- cated in ACC pathogenesis. The protein expression output of DTL is commonly referred to as CDT2. Since the
A
B
C
| Group | Cases | DTL | ||||
|---|---|---|---|---|---|---|
| - | + | ++ | +++ | Positive Rate (%) | ||
| Carcinoma#* | 18 | 2 (11.11) | 12 (66.67) | 2 (11.11) | 2 (11.11) | 88.89 |
| Adenoma 0 | 31 | 19 (61.3) | 12 (38.7) | 0 (0) | 0 (0) | 38.7 |
| Adjacent normal adrenal grand | 8 | 8(100.00) | 0 (0.00) | 0 (0.00) | 0 (0.00) | 0.00 |
Note: Negative and positive expression of DTL were compared using the Chi-square test, * Carcinoma vs. Normal: P=0.000; Fisher’s exact test, # Carcinoma vs. Adenoma: X’ = 11.708, P= 0.001; Fisher’s exact test, ” Adenoma vs. Normal: P = 0.042.
| Clinical features | N | DTL | |||||
|---|---|---|---|---|---|---|---|
| - | + | ++ | +++ | u | P value | ||
| Gender | |||||||
| Male | 19 | 8 | 8 | 2 | 1 | 269.50 | 0.729 |
| Female | 30 | 14 | 12 | 3 | 1 | ||
| Age | |||||||
| ≥ 42 years old | 23 | 11 | 8 | 3 | 1 | 294 | 0.913 |
| < 42 years old | 26 | 11 | 12 | 2 | 1 | ||
| Primary tumor (pT) | |||||||
| <5cm | 33 | 20 | 12 | 0 | 1 | 107.50 | 0.000 |
| ≥5cm | 16 | 2 | 8 | 5 | 1 | ||
| Infiltration metastasis | |||||||
| Yes | 7 | 1 | 3 | 2 | 1 | 74.50 | 0.024 |
| No | 42 | 21 | 17 | 3 | 1 | ||
| Clinical stage | |||||||
| I,II | 9 | 0 | 5 | 3 | 1 | 31.5 | 0.39 |
| III,IV | 9 | 2 | 4 | 2 | 1 | ||
Overall survival (n=18)
High Expression of DTL
1.0
Low Expression of DTL
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Survival
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p=0.04
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40
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function of free CDT2 is not yet clear, most of the current re- search has focused on studying the bound form of CDT2, that is, the CUL4-based E3 ubiquitin ligase CRL4CDT2 [17]. The CRL4CDT2 ubiquitin ligase complex, also known as DCX (DTL), is unique among ubiquitin ligases as its func- tion is related to DNA synthesis [17]. It ubiquitinates key cel- lular substrates, leading to their subsequent proteasomal degradation during the S-phase of the cell cycle in response to DNA damage [18]. CRL4CDT2 is composed of Cullin 4 (CUL4A or CUL4B), which acts as a scaffold protein; DNA Damage Binding Protein 1 (DDB1), which functions as an adaptor; and CDT2, which serves as the substrate receptor, playing a crucial role in determining substrate specificity [10]. CDT2 is the most active and key component of the E3 ubiquitin ligase CRL4CDT2. In recent years, studies have shown that the E3 ubiquitin ligase CRL4CDT2 can regulate a variety of substrates that play a key role in the physiologi- cal cell cycle and DNA damage response [17]. Given its cen- tral role in tumor progression, CRL4CDT2-mediated DTL overexpression may contribute to ACC malignancy by pro- moting uncontrolled cell proliferation and genomic instabili- ty. Targeting this ubiquitin ligase pathway could serve as a potential therapeutic strategy for ACC.
DTL, also known as L2DTL (Lethal(2) denticleless pro- tein homolog), is a human ortholog of the Drosophila lethal(2) denticleless gene (l(2)dt1). The gene was named based on its role in Drosophila development, where homozy- gous mutations result in embryonic lethality due to the absence of the ventral dentate band [19]. Bioinformatics analysis has identified that human L2DTL as an ortholog of Drosophila 1(2)dtl, encoding the WD40 repeat protein [20], which contains a Nuclear Localization Signal (NLS), a po- tential PEST (proline [P], glutamic acid [E], serine [S] and threonine [T]) sequence [21], a KEN box signal, and two D- box signals. Mitotic cyclins, PTTG1/Securin, and other cell cycle regulators, which are targeted for degradation during
cell cycle progression, have similar protein characteristics [22, 23]. Similar to Drosophila 1(2)dt1, human L2DTL is highly expressed in multiple fetal tissues, indicating its in- volvement in embryonic development. Studies on L2DTL knockout mice have demonstrated that its loss leads to early embryonic lethality [24]. These findings suggest that L2DTL is involved in cell cycle regulation and plays an important role in cell proliferation during embryonic development.
DTL plays a crucial role in maintaining genomic stabili- ty, primarily through its function within the CRL4CDT2 E3 ubiquitin ligase complex. DTL knockdown has been shown to consistently result in significant replication stress and ge- nomic instability [25]. Conversely, DTL overexpression leads to a decrease in substrate levels of its target proteins, leading to cell cycle dysregulation and contributing to patho- logical conditions, including tumorigenesis [26]. Studies have evidenced the importance of precise regulation of CR- L4CDT2 activity, as abnormal regulation of DTL has been observed in a variety of tumors [27] and DTL has recently been identified as a target for oncogenic viruses [28]. Studies have also shown that the expression of DTL is in- creased in invasive hepatocellular carcinoma (HCC), and its levels are positively correlated with tumor grade and a high mortality rate [24] underscoring its potential as a biomarker for aggressive cancers.
In order to study DTL protein expression in adrenocorti- cal tumors, immunohistochemical analysis on ACC, ACA, and adjacent normal tissues was performed. The result re- garding DTL localization was that the protein showed brown nuclear staining in both ACC and ACA tissues, which was consistent with the findings of Pan et al [24]. The expression rates of DTL were 88.89% in ACC, 38.7% in ACA, and almost no detectable expression in normal adreno- cortical tissues adjacent to the tumor.
The trend was that DTL expression increased progres- sively from normal adrenocortical tissues adjacent to the tu- mor to ACA to ACC, suggesting a strong association be- tween DTL upregulation and adrenocortical tumor progres- sion. The oncogenic role of DTL overexpression has been re- ported in gastric cancer (GC), For instance, Li et al [29] found that DTL was overexpressed in GC tissues when com- pared with adjacent normal tissues. Compared with normal gastric mucosal cells, seven gastric cancer cell lines overex- pressed DTL. Kobayashi et al [30] detected DTL protein overexpression in 57% (4/7) of GC cell lines and 42% (42/100) of primary GC tumor samples (42/100 cases; 42%). DTL downregulation inhibited cell proliferation, migration, and invasion in GC cell lines, suggesting its role in tumor ag- gressiveness. These findings suggest that DTL may serve as a key oncogene in multiple malignancies, including ACC and GC, making it a potential therapeutic target for cancer treatment.
In another study, Pan et al. [24] reported that L2DTL was overexpressed in 59% of 270 cases of resected, unifocal primary liver cancer and in all four examined hepatocellular carcinoma cell lines (Hep3B, Huh7, HA22T, and SKHep-1), whereas its expression was rare (2%) in normal liver tissue.
Similarly, Ueki et al. [31] demonstrated the upregulation of DTL/RAMP in the majority of breast cancer cases and across all breast cancer cell lines that they examined using semi- quantitative RT-PCR and Northern blot analysis. Moreover, silencing DTL/RAMP expression in T47D and HBC4 breast cancer cells via small interfering RNA effectively inhibited its expression and induced G2/M phase arrest, thereby in- hibiting cancer cell proliferation. These findings collectively indicate that DTL is frequently overexpressed in various ma- lignancies, highlighting its potential role in tumorigenesis. The findings of our study are consistent with these observa- tions.
Additionally, in this study, there was no significant corre- lation between DTL expression and clinicopathological fea- tures, such as gender, age, or clinical stage of adrenocortical tumors. However, DTL expression was significantly associat- ed with tumor size, invasion, and metastasis. Patients with ACC in the high DTL expression group had a significantly longer survival time when compared to those in the low DTL expression group.
Kobayashi et al. [30] found that DTL overexpression in gastric cancer was significantly linked to lymph node inva- sion, deeper tumor infiltration, and higher recurrence rates. Similarly, Pan et al. [24] observed that elevated expression of DTL in HCC was associated with high alpha-fetoprotein (AFP) levels, larger tumor diameter (> 5 cm), and advanced clinical stage. They also found that patients with HCC and high DTL expression had a lower ten-year cumulative survi- val rate than those with HCC and low DTL levels. Further- more, patients with HCC who had both high DTL expression and a P53 mutation had a worse prognosis. Functional studies showed that the knockdown of DTL significantly re- duced the invasion potential of HeLa and HA22T cells [24] suggesting that DTL overexpression could promote tumor cell invasiveness. The findings in our present study align with these results.
The DTL gene is evolutionarily conserved from nema- todes to humans. Under both normal and stress conditions, the CUL4-based E3 ligase (CRL4) regulates the degradation of DTL by targeting key substrates, including the replication licensing factor (CDT1), the cell cycle control protein (p21), and the chromatin modification factor (SET8). Through th- ese interactions, DTL plays a fundamental role in the regula- tion of the cell cycle. The precise mechanisms by which Cdt2 overexpression drives tumorigenesis are not fully un- derstood. However, one proposed mechanism involves the ability of the CRL4Cdt2 complex to promote cell cycle pro- gression by reducing the basal steady-state levels of the cell cycle inhibitor p21, thereby impairing p53-mediated DNA damage repair mechanisms [32, 33]. In addition, DTL has been shown to play a key role in promoting tumor cell prolif- eration, migration, and invasion through both p53-dependent and p53-independent pathways [30].
CONCLUSION
In conclusion, DTL shows promise as a novel biomarker for distinguishing between benign and malignant adrenocor-
tical tumors and for prognostic assessment in ACC. Our find- ings suggest that DTL could contribute to tumorigenesis in ACC, playing a role in its occurrence and progression. How- ever, this study is based on a limited sample size, necessitat- ing further validation through larger samples and additional tissue-based polymerase chain reaction and western blot to ensure more accurate results. In future research, we plan to explore the relationship between DTL and ACC cells, focus- ing on the specific molecular and cellular mechanisms through which DTL promotes tumor proliferation, invasion, and metastasis. This can provide new therapeutic targets for the diagnosis, treatment, and prognosis of ACC, as well as offer new insights for developing drugs for treating this ma- lignancy.
LIST OF ABBREVIATION
ACC = Adrenocortical Carcinoma
ACA = Adrenocortical Adenoma
GEO = Gene Expression Omnibus
DTL = Denticleless E3 ubiquitin Protein Ligase
AUTHORS’ CONTRIBUTIONS
The authors confirm their contribution to the paper as fol- lows: study conception and design: LXP, LL; data collec- tion: XY; analysis and interpretation of results: XL, SC, XHL; draft manuscript: DCL, ZJL. All authors reviewed the results and approved the final version of the manuscript.
ETHICS APPROVAL AND CONSENT TO PARTICI- PATE
The study was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (Ap- proval Number: 2025-E0233).
HUMAN AND ANIMAL RIGHTS
No animals were used in this research. All procedures performed in studies involving human participants were un- der the ethical standards of the institutional and/or research committee and with the 1975 Declaration of Helsinki, as re- vised in 2013.
CONSENT FOR PUBLICATION
All participants provided written informed consent for their involvement in the study. In cases where participants were under the age of 18, informed consent was additionally obtained from their parents or legal guardians.
AVAILABILITY OF DATA AND MATERIALS
The data that support the findings of this study are avail- able from the corresponding author upon reasonable request.
FUNDING
National Natural Science Foundation of China (No. 82260159).
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or otherwise.
ACKNOWLEDGEMENTS
We are particularly grateful to all the people who have given us help with our article.
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