Over-expression of Anillin Actin Binding Protein in Adrenocortical Carcinoma Tissues Is Associated With Poorer Prognosis of Patients
SHIN-NOSUKE YAMASHITA1,2*, YOSHIATSU TANAKA1,2*, SHAJEDUL ISLAM1,3, TAKAO KITAGAWA1, KAZUHIRO TOKUDA4, DURGA PAUDEL1, SARITA GIRI1, TOHRU OHTA1, FUMIYA HARADA2, HIROKI NAGAYASU2 and YASUHIRO KURAMITSU1,5
“Advanced Research Promotion Centre, 2Division of Oral and Maxillofacial Surgery, School of Dentistry, Health Sciences, and 5School of Medical Technology, University of Hokkaido, Ishikari-Tobetsu, Japan;
3Department of Immunology, The University of Texas MD Anderson Cancer center, Houston, TX, U.S.A .; 4Graduate School of Health and Welfare, Yamaguchi Prefectural University, Yamaguchi, Japan
Abstract
Background/Aim: Adrenocortical carcinoma (ACC) is a rare adrenal cortex cancer (0.7-2.0 cases per million) with high malignancy and poor prognosis (5-year survival <40%). Early detection is difficult due to adrenal location, and effective treatments are lacking, highlighting the need for prognostic biomarkers and therapeutic targets. Anillin actin-binding protein (ANLN), a key regulator of cytokinesis, is overexpressed in many cancers compared with normal tissues and is associated with poor prognosis and advanced stage. However, the clinicopathological significance of ANLN in ACC remains unclear.
Materials and Methods: ANLN mRNA expression and survival in patients with ACC [normal (n=128), stage I (n=9), stage II (n=37), stage III (n=16), stage IV (n=15)] were analyzed from TCGA database by using the UALCAN and GEPIA platforms, assessing ANLN mRNA expression across disease stages and its correlation with patient survival.
Results: ANLN was found to be significantly increased in stage IV ACC tissues compared with stage I, II and III ACC tissues (p<0.05). Furthermore, increased expression was significantly correlated with poor patient prognosis (p<0.005). Conclusion: ANLN may be a prognostic biomarker for patients with ACC and may play a role in tumor biology. Further studies are needed to determine whether ANLN is clinically useful as a prognostic factor in the treatment of ACC and to clarify its involvement in the mechanism of malignant progression of ACC.
Keywords: ANLN, adrenocortical carcinoma, Kaplan-Meier survival plot, TCGA, UALCAN, GEPIA.
Introduction
Adrenocortical carcinoma (ACC) is a rare tumor originating from the adrenal cortex, with an annual
incidence of 0.7-2.0 cases per million (1). The prognosis of ACC is very poor, with a 5-year survival rate of 38.6% after tumor removal surgery, with a median survival of 31.9 months. Furthermore, the development of metastasis
*These Authors contributed equally to this work.
☒ Yasuhiro Kuramitsu, MD, PHD, 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
Received November 23, 2025 | Revised December 4, 2025 | Accepted December 5, 2025
reduces the 5-year survival rate to less than 15% (2, 3). Therefore, to improve the prognosis of patients with ACC, it is important to select suitable prognostic biomarkers for treatment, but currently there are no clinically useful prognostic biomarkers for ACC that can benefit patients.
To identify molecules related to the prognosis of ACC, we used the TCGA database to search for molecules whose expression is significantly elevated in ACC tissue and that may significantly shorten patient survival. We previously reported that the expression of DDX39 and STMN1 is significantly elevated in ACC tissue, which significantly worsens patient prognosis (4, 5). The Cancer Genome Atlas (TCGA) is a database for molecular analysis. It contains over 20,000 primary cancer samples and their corresponding normal tumor samples for 33 different cancer types. It is available to the public free of charge to aid in cancer diagnosis, treatment, and research (6). To analyze the data in this cancer patient database, we used two platforms: the University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) and Gene Expression Profiling Interactive Analysis (GEPIA). UALCAN is a web-based tool for analyzing cancer OMICS data, allowing users to identify biomarkers and perform in silico validation of genes of interest (7). GEPIA is also a web-based tool with customizable features that allow users to identify biomarkers and perform in silico validation of genes of interest (8). We previously analyzed the TCGA database of pancreatic cancer patients using UALCAN and GEPIA platforms, and reported that anillin- actin binding protein (ANLN) mRNA expression was increased in pancreatic cancer tissues, and that patients with increased ANLN expression had a poor prognosis (9).
ANLN is a protein that binds to actin and is involved in cell division. It has been reported that its expression is elevated in many types of cancer tissues and cancer cells, and it is suspected to be related to the malignant progression, such as carcinogenesis, invasion into surrounding tissues, and metastasis to distant organs (10-16). Because ACC, like pancreatic cancer, is prone to metastasis and has a poor prognosis, we focused on ANLN as a prognostic factor for ACC.
We found that ANLN is more highly expressed in tissues from ACC patients than in normal adrenal cortex tissues, and that it is a prognostic factor that significantly worsens the prognosis of patients with ACC, i.e., significantly shortens their survival time.
Materials and Methods
Assessment of ANLN mRNA expression in cancer tissues from patients with different stages of adrenocortical carcinoma. The expression levels of ANLN mRNA in cancer tissues from patients with various stages of ACC registered in the TCGA database were investigated by using the UALCAN platform, a web server for cancer and normal gene expression profiling [stage 1 (n=9), stage 2 (n=37), stage 3 (n=16), stage 4 (n=15)] (7), The gene name “ANLN” was registered in the TCGA database .. By using the GEPIA platform, another web server for cancer and normal gene expression profiling, the expression levels of ANLN mRNA in ACC tissues and normal adrenal cortex tissues were examined [ACC tissues (n=77) vs. normal adrenocortical tissues (n=128)] (8). The gene name “ANLN” was entered into the TCGA database and selected DIY Boxplot (cancer vs. normal tissue analysis) to obtain the results. This analysis showed a series of ACC studies and the expression of ANLN in cancer and normal tissues. The filters were set as follows: i) Gene: ANLN, ii) Threshold settings: Log2FC cutoff 2 with p-value of <0.05.
Survival analysis according to ANLN mRNA expression levels in adrenocortical carcinoma tissues. The effect of high or low ANLN expression levels in ACC tissues on the survival of patients with ACC was investigated by using the GEPIA platform. The gene name “ANLN” was entered into the TCGA database, and the median cutoff value was selected to generate Kaplan-Meier curves for patients with ACC. Furthermore, we used the bioinformatics platform ULCAN to investigate the effect of ANLN mRNA expression levels on the survival of patients with ACC. A p-value <0.05 was considered statistically significant.
A
B
6
*
Expression of ANLN in ACC based on individual cancer stages
50
p=2.083500E-03
p=5.177300E-03
5
p=2.178100E-02
40
Transcript per million
4
30
3
..
20
2
10
0
1
Stage 1 (n=9)
Stage 2 (n=37)
Stage 3 (n=16)
Stage 4 (n=15)
TCGA samples
0
ACC (num(T)=77; num(N)=128)
Results
ANLN mRNA expression was up-regulated in adrenocortical carcinoma tissues from stage IV patients compared with stage I, II and III patients. To investigate whether the increased ANLN mRNA expression level in ACC tissues depends on the stage of patients with ACC, the TCGA dataset was analyzed by using the UALCAN platform. Figure 1A shows a graph of ANLN mRNA expression in ACC tissues based on individual tumor stage. ANLN mRNA expression was significantly increased in tumor tissues from stage IV patients compared with stage I, II and III patients (p<0.01,p<0.01, p<0.05, respectively).
ANLN mRNA expression was higher in ACC tissues compared with normal adrenal cortex tissues. TCGA datasets were
analyzed by using the GEPIA platform to examine ANLN mRNA expression in ACC tissues. ANLN mRNA levels were higher in ACC tissues (n=77) compared with normal adrenal cortex tissues (n=128) (p<0.05) (Figure 1B).
High expression levels of ANLN are correlated with poorer prognosis in patients with adrenocortical carcinoma. Kaplan-Meier survival plots were generated for patients with ACC with high ANLN expression and patients with ACC with low/moderate ANLN expression by using the UALCAN platform. Increased expression levels of ANLN mRNA correlated with shorter patient survival (p=0.0017) (Figure 2). Additionally, overall survival and disease- free status were analyzed by using the GEPIA platform. Kaplan-Meier survival plots showed that increased expression levels of ANLN mRNA were found to correlate
Effect of ANLN expression level on ACC patient survival
1.00
0.75
Survival probability
0.50
0.25
p=0.0017
Expression level
High expression (n=20)
Low/Medium expression (n=59)
0.00
0
1,000
2,000
3,000
4,000
Time (days)
with shorter overall (p<0.01) (Figure 3A) and disease-free survival of patients with ACC (p=0.0033) (Figure 3B).
Discussion
In the present study, ANLN mRNA expression and Kaplan- Meier survival in patients with ACC were analyzed by using the bioinformatics platforms UALCAN and GEPIA. ANLN mRNA expression levels were significantly increased in ACC tissues from stage IV patients compared with those from stage I, II and III patients with ACC. Additionally ANLN mRNA levels were higher in ACC tissues compared with normal adrenal cortex tissues. Furthermore, increased
expression of ANLN was found to correlate with shorter overall and disease-free survival in patients with ACC. These results suggest that ANLN is up-regulated in ACC tissues, and that this expression adversely affects the prognosis of patients with ACC.
ACC is rare but highly aggressive in nature, with a high invasive and metastatic potential. Although it is unclear what drives the aggressive malignant progression of ACC, several molecules and signaling pathways have been reported as candidates associated with the malignant phenotype of ACC. Zon et al. identified centromere protein M (CENPM) as a driver gene promoting the high metastatic potential of ACC. CENPM is a key gene that promotes ACC
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Overall survival
Disease-free survival
1.0
Low.ANLN.T.PM
1.0
High ANLN TPM
Low ANLN TPM
High ANLN TPM
Logrank p=8.3e-05
Logrank p=0. 0033
0.8
HR(high)=5.3
p(HR)=0.00039
0.8
HR(high)=2.7
n(high)=38
p(HR)=0.0046
Percent survival
n(high)=38
0.6
n(low)=38
Percent survival
0.6
n(low)=38
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0.2
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0
50
100
150
0
50
100
150
Time (months)
Time (months)
metastasis and is a powerful prognostic biomarker for ACC. Furthermore, the immune checkpoint ligand FGL1 is over-expressed in ACC and promotes ACC metastasis. Silencing CENPM inhibits ACC metastasis in vitro and in vivo through physical interaction with FGL1 (17). Zhou et al. reported that kinesin family member 11 (KIF11) expression was up-regulated in ACC tissues, and that increased KIF11 expression was significantly associated with shorter overall survival, disease-specific survival and progression- free survival. Furthermore, they confirmed that a specific KIF11 inhibitor significantly inhibited the proliferation and invasion of ACC cells in vitro (18). Huang et al. reported that endothelial cell-specific molecule 1 (ESM1) over- expression was significantly observed in patients with ACC and that this ESM1 over-expression was significantly correlated with decreased overall survival in ACC patients. Furthermore, they demonstrated that ESM1 promotes the G2/M transition of mitosis and cell proliferation via the DLL4-Notch signaling pathway (19). In contrast, Chen et al. reported that the level of fibronectin type III domain-
containing protein 5 (FNDC5) in ACC tissues was reduced compared with normal tissues, and that over-expression of FNDC5 in vitro inhibited ACC cell proliferation, migration, and invasion and promoted cell apoptosis, suggesting that over-expression of FNDC5 activated the AMPK/ mTOR signaling pathway, inhibiting ACC cell proliferation, migration, and invasion and promoting apoptosis (20). The purpose of this study was to analyze the TCGA database using the UALCAN and GEPIA platforms to identify previously unreported molecules involved in the malignant progression of ACC. We focused on ANLN, which is highly associated with the prognosis of pancreatic cancer, which has a worse prognosis than ACC (9).
Pancreatic cancer is one of the most metastatic cancers with poor prognosis. We have reported that ANLN expression is significantly higher in pancreatic cancer tissues than in normal tissues, and that patients with high expression of ANLN in pancreatic cancer tissues have a poor prognosis. ANLN is an actin-binding protein which plays important roles in cytokinesis (10).
Over-expression of ANLN mRNA has been reported not only in pancreatic cancer but also in various other cancer types, and is widely involved in the progression and metastasis of many cancer types (11-16). This suggests that ANLN expression is elevated in most types of cancer tissues compared with normal tissues, and elevated ANLN expression in these cancer tissues is closely correlated with poor prognosis and cancer cell progression (11-16, 21).
Previous reports suggest that ANLN is involved in the malignant progression of cancer cells by activating several pathways. It has been suggested that the PI3K/AKT signaling pathway may be activated by ANLN, promoting proliferation, migration, and invasion (22). ANLN has also been reported to stabilize c-Myc and activate the MAPK signaling pathway in cancer cells (23). Furthermore, it has been reported that ANLN functions as an oncogene in cancer cells by activating the JNK signaling pathway (14). Although the detailed mechanism of activation of these pathways by ANLN is still unclear, there is no doubt that ANLN functions as a promoter of malignant progression of cancer cells. However, there have been no reports of ANLN in ACC cells. Using TCGA, we analyzed the relationship between ANLN expression and the prognosis of patients with ACC, and found that patients with significantly higher ANLN expression in cancer tissues had a poor prognosis. Therefore, it is necessary to elucidate the mechanism by which ANLN promotes the malignant progression of cancer in ACC cells.
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 conception and design of the study. Data collection and analysis were performed by Shin-nosuke Yamashita, Yoshiatsu Tanaka, Shajedul Islam and Yasuhiro Kuramitsu. Shin-nosuke Yamashita and
Yoshiatsu Tanaka wrote the first draft of the manuscript, Takao Kitagawa and Yasuhiro Kuramitsu commented on an earlier version of the manuscript. All Authors read and approved the final manuscript.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
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