Up-regulated DDX39 in Adrenocortical Carcinoma Is Associated With Patient Survival
YOSHIATSU TANAKA1*, SHIN-NOSUKE YAMASHITA1*, SHAJEDUL ISLAM1,
TAKAO KITAGAWA1, KAZUHIRO TOKUDA2, DURGA PAUDEL1, SARITA GIRI1,
TOHRU OHTA1, FUMIYA HARADA3, HIROKI NAGAYASU3 and YASUHIRO KURAMITSU1,4
1 Advanced Research Promotion Centre, 3Division of Oral and Maxillofacial Surgery School of Dentistry, and
4School of Medical Technology, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Japan;
2Graduate School of Health and Welfare, Yamaguchi Prefectural University, Yamaguchi, Japan
Abstract. Background/Aim: Adrenocortical carcinoma is a very rare tumor characterized by poor prognosis and high mortality. The origin of this tumor is primarily the adrenal cortex. The 5-year overall survival rate of patients with adrenocortical carcinoma has not improved despite therapeutic advances. Early detection of this malignancy remains difficult, and no standard curative therapy currently exists. Therefore, it is important to understand the biology of adrenocortical carcinoma, and to identify prognostic biomarkers and molecular targets for its therapy. DDX39 is an Asp-Glu-Ala- Asp (DEAD)-box RNA helicase, which is required for transcription, splicing and transport of mRNA. There are some reports about overexpression of DDX39 in tumor tissues and cells (lung squamous cell cancer, gastrointestinal stromal tumor, urinary bladder cancer, malignant pleural mesothelioma). However, the clinicopathological involvement of DDX39 in adrenocortical carcinoma has not yet been documented. Materials and Methods: The GEPIA, GEPIA2, and UALCAN platforms were used to analyze DDX39 mRNA expression and survival in patients with adrenocortical carcinoma. Results: DDX39 was found to be significantly up-regulated in adrenocortical carcinoma tissues, and this up-regulation inversely correlated with prolonged patient survival. Conclusion: DDX39 may be a potential prognostic biomarker in patients with adrenocortical carcinoma.
*These Authors contributed equally to this work.
Correspondence to: Yasuhiro Kuramitsu, MD, Ph.D., Advanced Research Promotion Center, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan. Tel: +81 133231630, e-mail: climates@hoku-iryo-u.ac.jp
Key Words: DDX39, adrenocortical carcinoma, Kaplan-Meier survival plot.
Adrenocortical carcinoma is a rare tumor derived from the adrenal cortex. The annual incidence is 0.7-2.0 individuals per million (1). Although this carcinoma occurs at any age, patients younger than 5 years old and 40-50 years old are in majority (2, 3). This cancer shows poor prognosis. Overall 5-year survival of the patients following surgery to remove a tumor was 38.6%, and median survival period was 31.9 months (4). However, so far there is no clinically useful prognostic biomarker for adrenocortical carcinoma.
DDX39 is an Asp-Glu-Ala-Asp (DEAD)-box RNA helicase. It unwinds double-stranded RNA molecules, and also plays roles in transcription, splicing, RNA transport, ribosome biogenesis, RNA editing, RNA decay and translation. Furthermore, DDX39 is very important for telomere protection and maintenance (5-7). The roles of DDX39 in cancer have been reported in human lung squamous cell carcinoma tissues (8), gastrointestinal stromal tumor tissues from patients with poor prognosis (9), urinary bladder cancer tissues (10) human malignant pleural mesothelioma cells (11) and human gemcitabine-resistant pancreatic cancer cells (12).
Although Kikuta et al. reported DDX39 as a poor prognostic biomarker in patients with gastrointestinal stromal (9), Kato et al. showed that DDX39 suppressed invasion of bladder cancer cells (10). Therefore, the effects of DDX39 on the prognosis of patients of various cancers are still unclear. Furthermore, the clinicopathological relevance of DDX39 expression in adrenocortical carcinoma has not been yet documented. This study aimed to clarify the clinical significance of DDX39 expression in adrenocortical carcinoma patients by using several bioinformatics platforms.
Materials and Methods
mRNA expression of DDX39 in adrenocortical carcinoma tissues. To explore DDX39 mRNA expression levels in adrenocortical carcinoma tissues, the GEPIA, a web server for cancer and normal
gene expression profiling (13) was used. In the TCGA database, the gene name “DDX39A” was entered. The expression DIY Boxplot (cancer vs. normal analysis) was selected to retrieve the results. This analysis presented a series of adrenocortical carcinoma studies and related DDX39A expression in cancer and normal tissues. The filters were set as follows: i) Gene: DDX39A, ii) Threshold settings: Log2FC cutoff 2 with p-value of <0.05.
Evaluation of DDX39 expression in cancerous tissues of patients with different stages of adrenocortical carcinoma. In the TCGA database, the gene name “DDX39” was entered. DDX39 mRNA expression levels in cancerous tissues from patients with different stages of adrenocortical carcinoma were explored from the TCGA databases, using the UALCAN platform.
Survival analysis according to mRNA expression levels of DDX39 in adrenocortical carcinoma tissues. The survival analysis was performed to investigate the DDX39A expression in adrenocortical carcinoma by using the GEPIA2 platform (14). In the GEPIA2 database, the gene name “DDX39A” was entered, and median cutoff was selected to generate Kaplan-Meier curves for patients with adrenocortical carcinoma. Moreover, ULCAN a bioinformatics platform was used to confirm the impact of mRNA expression levels of DDX39A on the survival of patients with adrenocortical carcinoma. A p-value of <0.05 was considered statistically significant.
Results
DDX39 mRNA expression was increased in adrenocortical carcinoma tissues. To determine DDX39A mRNA expression in adrenocortical carcinoma tissues, TCGA datasets were analyzed by using the GEPIA platform. The results demonstrated that DDX39A mRNA levels were up-regulated in adrenocortical carcinoma tissues (n=77) compared to normal tissues (n=128) (p<0.05) (Figure 1). We then analyzed whether the increased DDX39A expression levels were dependent on the stage of adrenocortical carcinoma. Figure 2 shows the expression of DDX39 in adrenocortical carcinoma based on individual cancer stages. The expression of DDX39A increased progressively with the stages of adrenocortical carcinoma.
High levels of DDX39A expression are inversely correlated with prolonged patient survival in adrenocortical carcinoma. The Kaplan-Meier survival plot was generated using the GEPIA2 platform, and the overall survival status was analyzed. Increased expression levels of DDX39A were found to be inversely correlated with prolonged patient survival (p=0.00000057) (Figure 3A). In addition, the increased expression levels of DDX39A were inversely correlated with the disease-free state of patients (p=0.0000038) (Figure 3B). In addition, we also used UALCAN platform and generated the Kaplan-Meier survival plot of the patients with DDX39- up-regulated and down-regulated adrenocortical carcinoma tissues. The results showed that increased expression levels of DDX39 were inversely correlated with prolonged patient survival (p<0.0001) (Figure 4).
8
::
6
4
2
0
ACC (num(T)=77; num(N)=128)
Discussion
In the present study, the expression of DDX39 mRNA and Kaplan-Meier survival were analyzed in patients with adrenocortical carcinoma by using bioinformatics platforms of GEPIA, GEPIA2 and UALCAN. The mRNA expression level of DDX39 was significantly up-regulated in adrenocortical carcinoma tissues compared to normal tissues, and this up-regulated DDX39 expression was inversely correlated with prolonged patient survival.
Analysis using GEPIA and UALCAN revealed that DDX39 mRNA expression is high in adrenocortical carcinoma tissues, but is RNA expression also high in other cancers? Wang et al. reported that DDX39 expression was higher in ER-positive
Expression of DDX39 in ACC based on individual cancer stages
250
200
Transcript per million
150
100
50
0
Stage 1 (n=9)
Stage2 (n=37)
Stage 3 (n=16)
Stage4 (n=15)
TCGA samples
A
Overall Survival
B
Disease Free Survival
1.0
Low.DDX39A .TPM
1.0
High DDX39A TPM
Low DDX39A Group
High DDX39A Group
Logrank p=5.7e-07
0.8
HR(high)=8.8
Logrank p=3.8e-06
p(HR)=1.9e-05
0.8
HR(high)=5.1
Percent survival
n(high)=38 n(low)=38
p(HR)=2.9e-05
Percent survival
n(high)=38 n(low)=38
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
0
50
100
150
0
50
100
150
Months
Months
Effect of DDX39 expression level on ACC patient survival
1.00
0.75
Survival probability
0.50
0.25
p<0.0001
Expression Level +High expression (n=20) +Low/Medium-expression (n=59)
0.00
0
1,000
2,000
3,000
4,000
Time (days)
breast cancer tissues than in normal breast tissues, and that the degree of high expression was significantly related to the size of the cancer tissue and the degree of poor differentiation (15). Naboulsi et al. reported that the amount of DDX39 protein in hepatocellular carcinoma tissues was higher than in normal liver tissues (16). On the other hand, Ma et al. reported that DDX39 expression was lower in colorectal cancer tissues than in normal tissues (17).
Is DDX39 related to survival in other cancers as well? Bao et al. reported that patients with high DDX39 expression in clear cell renal cancer had shorter overall survival and progression-free interval (18). Wang et al. reported that increased expression of DDX39 was significantly associated with poor outcomes in patients with ER-positive breast cancer (15). Kikuta et al. showed poor prognosis of patients with high DDX39 expressing gastrointestinal stromal tumor (9). Conversely, Ma et al. reported that patients with colorectal cancer tissues with low DDX39 expression had a poor prognosis (17). Considering these previous studies, the
expression of DDX39 and its effect on prognosis may differ depending on the cancer type. Further research is needed.
It is unclear why DDX39 worsens the prognosis of patients with adrenocortical carcinoma. DDX39 is an RNA helicase that unwinds double-stranded RNA molecules and plays important roles in transcription, splicing, RNA transport, ribosome biogenesis, RNA editing, RNA decay and translation, and may therefore be essential for the survival and proliferation of cancer cells. Wang et al. showed that down-regulation of DDX39 by siRNA significantly reduced the cell growth and invasion ability of the human breast adenocarcinoma cell line MCF-7 cells (15). However, there are almost no reports on the mechanism by which DDX39 worsens the prognosis of patients with cancer. Further studies of DDX39 in cancer cells are needed.
Although the present study suggested that high expression of DDX39 may be related to the poor prognosis of patients with adrenocortical carcinoma, there have been no report on whether it is possible to detect DDX39 in human serum to
determine the prognosis, nor on whether autoantibodies against adrenocortical carcinoma cells exist in the serum. If it were possible to quantitatively detect DDX39 or anti- DX39 antibodies in the serum of patients with adrenocortical carcinoma, this would help to predict their prognosis.
Further studies are needed to clarify the exact mechanisms by which DDX39 promotes the progression of adrenocortical carcinoma.
Conflicts of Interest
The Authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Authors’ Contributions
All Authors contributed to the study’s conception and design. Data collection and analysis were performed by Yoshiatsu Tanaka, Shin- nosuke Yamashita, Takao Kitagawa, Shajedul Islam and Yasuhiro Kuramitsu. Yoshiatsu Tanaka, Shin-nosuke Yamashita and Takao Kitagawa wrote the first draft of the manuscript, Shajedul Islam and Yasuhiro Kuramitsu commented on previous versions of the manuscript. All Authors read and approved the final manuscript.
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Received December 24, 2024 Revised January 9, 2025 Accepted January 10, 2025