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RESEARCH PAPER
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Homeobox A5 activates p53 pathway to inhibit proliferation and promote apoptosis of adrenocortical carcinoma cells by inducing Aldo-Keto reductase family 1 member B10 expression
Danyan Chenª, Zhaonan Shenb, Xi Chengc, Qi Wangd, Junlin Zhoue, Fang Renf, Yue Sung, Hongman Wanga, and Rongxi Huang İD a
aDepartments of Endocrinology, Chongqing General Hospital, University of Chinese Academy of Sciences, Chongqing, China; bDepartments of Nephrology, The Fifth People’s Hospital of Chongqing, Chongqing China; “Departments of Science & Education, Chongqing General Hospital, University of Chinese Academy of Sciences, Chongqing, China; dDepartments of Laboratory, Chengdu Sixth People’s Hospital, Chengdu, Sichuan Province China; eDepartments of Endocrinology, The First Affiliated Hospital of University of South China, Hengyang, Hunan Province China; ‘Departments of Emergency, Chongqing General Hospital, University of Chinese Academy of Sciences, Chongqing China; 9Departments of Geriatrics, The First Affiliated Hospital of Chongqing Medical University, Chongqing China
ABSTRACT
Aldo-Keto Reductase Family 1 Member B10 (AKR1B10) and Homeobox A5 (HOXA5) are both down-regulated in adrenocortical carcinoma (ACC), and HOXA5 is predicted to bind to the promoter of AKR1B10. We aimed to investigate whether HOXA5 could bind to AKR1B10 to regulate ACC cells proliferation and apoptosis. The expression of AKR1B10 and HOXA5 in ACC patients and the relationship of their expression between ACC prognosis were evaluated by searching database. Then, NCI-H295R cells were overexpressed to detect the alteration of cell proliferation, apoptosis and the expression of p53 and p21 proteins. The interaction between AKR1B10 and HOXA5 was validated by luciferase report and chromatin immunoprecipitation. Finally, NCI-H295R cells were silenced with HOXA5 in the presence of AKR1B10 overexpression, and then cell proliferation and apoptosis were also assessed. Results revealed that AKR1B10 and HOXA5 are down-regulated in ACC patients and the low expression of it is correlated with low percent of overall survival (OS) and disease free survival (DFS). Compared with Y1 cells, SW- 13 and NCI-H295R cells exerted lower expression of AKR1B10 and HOXA5. AKR1B10 significantly inhibited cell viability, colony formation and expression of Ki67 and PCNA, but promoted apop- tosis and expression of p53 and p21 in NCI-H295R cells. HOXA5 could interact with AKR1B10 and enhance AKR1B10 expression. Furthermore, HOXA5 knockdown obviously blocked the effect of AKR1B10 overexpression on NCI-H295R cells proliferation and apoptosis. In conclusion, HOXA5 could bind to AKR1B10 promotor to increase its expression, activate p53 signaling, thereby inhibiting proliferation and promoting apoptosis of ACC cells.
ARTICLE HISTORY
Received 5 March 2021 Revised 25 April 2021 Accepted 27 April 2021
KEYWORDS
Adrenocortical carcinoma; Aldo-Keto reductase family 1 member b10; hoxa5 protein; human; hyperaldosteronism
HOXA5
AKR1B10
p53 pathway
proliferation
apoptosis
Adrenocortical carcinoma
CONTACT Rongxi Huang huangrongxirx@outlook.com The Chongqing General Hospital, University of Chinese Academy of Sciences, No. 118, Xingguang Avenue, Liangjiang New District, Chongqing 401147, China
☒ Supplemental data for this article can be accessed here.
Introduction
Adrenocortical carcinoma (ACC), a rare endocrine aggressive malignancy, occurs in the globular zone of the adrenal cortex which can secrete aldosterone [1,2]. The occurrence rate of ACC ranges from approximately 1 to 2 cases per million persons per year [3]. ACC carries a poor prognosis due to its tendency to metastasize before diagnosis and has a high risk of relapse post radical surgery [4]. Therefore, it is of great clinical significance to screen the differentially expressed genes of ACC and to explore the molecular mechanism of this disease.
Aldo-Keto Reductase Family 1 Member B10 (AKR1B10) is an oxidoreductase with NADPH (reduced nicotinamide adenine dinucleotide phos- phate) as a coenzyme, and can catalyze the con- version of aldehydes and ketones into corresponding alcohols [5]. Studies have found that AKR1B10 is highly expressed in liver cancer, breast cancer, lung cancer and other tumor tissues [6-8]. Nevertheless, it is reported that compared with adjacent normal colorectal tissues, AKR1B10 in colorectal cancer (CRC) tissue is down- regulated and is related to the patient’s clinic pathological conditions. Loss of AKR1B10 pro- motes the proliferation and migration of CRC cells in vitro [9]. In addition, the results in the Gene Expression Profiling Interactive Analysis (GEPIA) database also shows that the expression of AKR1B10 is significantly down-regulated in the tissues of patients with ACC, and is related to the poor survival of patients. However, whether AKR1B10 was down-regulated in ACC cells and played a role in the progression of ACC has not been illustrated.
Homeobox A5 (HOXA5) is a member of the HOX family, and its encoded protein is a transcription factor that is widely involved in the normal physiological and pathological pro- cesses of the human body via regulating human embryonic development and adult stem cell dif- ferentiation [10]. The role of HOXA5 in promot- ing or suppressing cancer in malignant tumors from different tissues has been gradually clarified. For example, the loss of HOXA5 in breast cancer cells can promote tumor cells to differentiate in the direction of cancer stem cells, thereby
promoting tumor progression [11]. In CRC, HOXA5 is down-regulated and its expression induces loss of the cancer stem cell phenotype, preventing tumor progression and metastasis by inhibiting wnt signaling [12]. Moreover, HOXA5 has been reported to cooperate with p53 to play a role in lung cancer mammary tumorigenesis [13,14]. Notably, HOXA5 is also down-regulated in ACC and it is predicted to bind to the promo- ter of AKR1B10 after searching JASPAR database. Therefore, we speculated that HOXA5 could bind to the promoter of AKR1B10 to modulate ACC progression.
In the present study, we aimed to investigate the expression of HOXA5 and AKR1B10 in ACC cell lines and to clarify whether HOXA5 could bind to the promoter of AKR1B10 and regulate its expres- sion, thereby modulating ACC cells proliferation and apoptosis through p53 signaling. Our findings might provide a novel therapeutic target for the treatment of ACC.
Materials and methods
Bioinformatics analysis
GEPIA is a time-saving and intuition web applica- tion that is used for gene expression analysis based on abundant data from TCGA and the GTEx databases. AKR1B10 and HOXA5 mRNA expres- sion in ACC tissues and normal adrenocortical samples was obtained with GEPIA. Additionally, the survival analysis with AKR1B10 or HOXA5 subgroup and the relationship between AKR1B10 or HOXA5 and clinicopathological information were also analyzed.
Cell culture and transfection
Human adrenal normal cell line (Y1) and ACC cell lines (SW-13, NCI-H295R) were obtained from ATCC (Manassas, USA) and cultured in DMEM (Gibco, USA) containing 10% fetal bovine serum (FBS) and 100 U/mL penicillin-100 µg/mL strep- tomycin (Beyotime, Shanghai, China) in a 5% CO2 incubator at 37°C.
Full-length cDNAs of human AKR1B10 (over- expression-AKR1B10, Oe-AKR1B10) or HOXA5 (Oe-HOXA5) were cloned into the pcDNA3.1
vector (Thermo Fisher Scientific, Inc.). A pcDNA3.1 empty vector was used as a negative control (Oe-NC). The short hairpin RNA (shRNA) against HOXA5 (shRNA-HOXA5) and negative control shRNA (shRNA-NC) were designed and synthesized by GeneScript (Nanjing, China). NCI- H295R cells in logarithmic growth phase were selected and seeded in the 6-well plates. Cell trans- fection was performed when the cell confluence was up to 50%-60% according to the instructions of Lipofectamine 2000 (Invitrogen, USA). Following 48 h of transfection, the cells were col- lected for subsequent experiments.
Cell counting kit-8 (CCK-8)
NCI-H295R cells that transfected with indicated vectors were cultured in 96-well plates with 2 × 103 per well. Subsequently, 10 ul CCK-8 reagent (Beyotime, China) was added to each well after cell culture for 24, 48 and 72 h, and incubated at 37°℃ for 2 h in the dark. The optical density of each well was measured at a wavelength of 450 nm using a microplate reader (Bio-Rad, USA).
Colony formation assay
NCI-H295R cells in the logarithmic growth phase were seeded for colony formation assay into dishes at a density of 200 cells per dish. The cell culture medium was replaced every 3 days and cell culture was terminated when macroscopic colonies could be observed. Colonies were then washed with PBS, fixed with 4% paraformaldehyde and stained with Gimsa solution for 10-30 min. The number of colonies with over 10 cells were photographed with a microscope at low magnification (x100).
Western blot
Total proteins from cells were extracted using RIPA lysis buffer (Beyotime Biotechnology, China) containing protease inhibitor (Roche, Switzerland). Protein samples were subjected to SDS-PAGE and then transferred onto PVDF membranes (Roche, Switzerland). After being blocked with 5% nonfat milk for 2 h at room temperature, the membranes were incubated with
primary antibodies (Abcam, UK; 1:1000 dilution) against AKR1B10, Ki67, PCNA, Bcl-2, Bax, cleaved-caspase3 (c-caspase3), pro-caspase3, c-cas- pase9, pro-caspase9, p53, p21, HOXA5 and GAPDH at 4℃ overnight. The membranes were then being incubated with a goat anti-mouse/rab- bit secondary antibody (Abcam, UK) for 2 h at room temperature. Enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc., USA) was used to visualize the protein bands in a Bio-Rad ChemiDoc XRS Imaging system (Hercules, USA).
Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was isolated from cells using TRIzol reagent (Thermo Fisher Scientific, Inc.) and then reversely transcripted into cDNA using PrimeScript Reverse Transcriptase (Takara, Japan) following the manufacturer’s instructions. The expression level of gene was measured using the SYBR-Green method, and products were detected by StepOnePlus™ Real-time PCR system (Applied Biosystems, USA). The primers used were as follow:
AKR1B10 (forward), 5’-
GACCCCTTGTGAGGAAAGCC-3’, AKR1B10
(reverse), 5’-
ATTGCAACACGTTACAGGCCC-3’; HOXA5 (forward), 5’-
ATCCAAATGGCCCGGACTAC-3’, HOXA5 (reverse) 5’- AGTCCCTGAATTGCTCGCTC-3’; GAPDH (forward) 5’-
CAACAGCCTCAAGATCATCAGC- 3’, GAPDH (reverse) 5’-TTCTAGACGGCAGGTCAGGTC-3’. GAPDH was used as reference gene, and the rela- tive gene expression levels were calculated using the 2-AACT analysis method.
Tunel staining
Tunel staining was utilized to observe cell apopto- sis through a Tunel Assay kit (Beyotime). Briefly, after being fixed with 4% paraformaldehyde for 30 min, cells were treated with 0.3% Triton X-100 at room temperature for 5 min. Thereafter, 50 uL Tunel detection solution was added to cells and incubated at 37℃ for 1 h in the dark. Apoptotic cells were observed under
a fluorescence microscope (Olympus Corporation, ×200) after mounting with an anti-fluorescence quenching mounting solution.
Dual-luciferase report
To verify interactions between HOXA5 and AKR1B10 promoter, wide type (WT) or mutant (MUT) AKR1B10 were cloned into the pGL4 luci- ferase reporter vectors, and then co-transfected with pcDNA3.1-HOXA5 (Oe-HOXA5) or NC (Oe-NC). At 48 h post-transfection, dual Luciferase Assay (Promega, USA) was applied to determine the luciferase reporter activities accord- ing to the manufacturer’s instructions.
Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was carried out according to a standard protocol. Briefly, after being crosslinked with 1% formaldehyde for 10 min at 37°℃, NCI- H295R cells were subjected to ChIP assay with a High-Sensitivity Kit (Abcam). The antibodies used in this assay included anti-HOXA5 and IgG (negative control). The primer sequences of AKR1B10 used for the qRT-PCR assay were: 5’- GACCCCTTGTGAGGAAAGCC-3’ (forward), 5’- ATTGCAACACGTTACAGGCCC-3’ (reverse).
Statistical analysis
All data were expressed as the mean ± standard deviation of from three independent experiments for each technique. Statistical analysis was conducted using GraphPad Prism software (version 6.0; GraphPad Software, Inc.). Differences among two groups were analyzed using Student’s test, among multiple groups were assessed using one-way analy- sis of variance (ANOVA) followed by Tukey’s test. P < 0.05 was considered statistically significant.
Results
AKR1B10 is down-regulated in ACC cell lines and overexpression of it inhibits NCI-H295R cells proliferation
A considerable body of evidence indicates that AKR1B10 participates in the occurrence and
development multiple cancer, such as nasophar- yngeal carcinoma, lung cancer and colorectal cancer [7-9]. After searching GEPIA database, we found that AKR1B10 is down-regulated in tissues of ACC patients (Figure 1a). Besides, low level of AKR1B10 is correlated with low overall survival (OS) and disease free survival (DFS), whereas high level of AKR1B10 predicts longer OS and DFS (Figure 1b and c) of ACC, indicating that AKR1B10 plays a beneficial role in inhibiting ACC. Thereafter, we measured the mRNA and protein expression of AKR1B10 in adrenal normal cell line (Y1) and ACC cell lines (SW-13, NCI-H295R). In accordance with the data from GEPIA, AKR1B10 was down- regulated in SW-13 and NCI-H295R cell lines, especially in NCI-H295R, compared with that in Y1 (Figure 2a and b). We then overexpressed AKR1B10 in NCI-H295R cells and results in Figure 2c and d demonstrated the significant increase in AKR1B10 expression. As shown in Figure 2e, AKR1B10 overexpression markedly reduced cell viability at 48 and 72 h. In addition, the colony formation of NCI-H295R cells was markedly inhibited upon AKR1B10 overexpres- sion (figure 2f). Consistently, the expression of proteins involved in cell proliferation including Ki67 and PCNA was also reduced by AKR1B10 overexpression (Figure 2g).
Overexpression of AKR1B10 induces apoptosis and p53 pathway activation of NCI-H295R cells
To study the effects of AKR1B10-upregulation on the apoptosis of ACC cells, the apoptosis of NCI- H295R cells was evaluated by Tunel staining and western blot assays. As demonstrated in Figure 3a, the number of Tunel-positive cells was highly increased after AKR1B10 overexpression, suggest- ing the occurrence of apoptosis. At the same time, Bcl-2 expression was reduced, whereas Bax, c-cas- pase3 and c-caspase9 expressions were remarkably enhanced upon AKR1B10 overexpression, indicat- ing the promotion of AKR1B10 on cell apoptosis (Figure 3b). Besides, AKR1B10 overexpression sig- nificantly up-regulated p53 and p51 expressions, revealing the activation of p53 signaling induced by AKR1B10 (Figure 3c).
HOXA5 is down-regulated in ACC cell lines and can bind to AKR1B10 to regulate AKR1B10 expression
To explore the possible regulatory mechanisms of AKR1B10 in the proliferation and apoptosis of ACC cells, the GEPIA database was employed to assess the expression of HOXA5 in adrenocortical tissues patients with ACC. After searching GEPIA database, we found that HOXA5 is notably down- regulated in ACC tissues (Figure 4a). Meanwhile, low level of HOXA5 is correlated with low OS, whereas high level of HOXA5 predicts longer OS (Figure 4b) of ACC, suggesting that HOXA5 may also play a beneficial role in inhibiting ACC. In addition, data from ENCORI reveals that the expression of HOXA5 and AKR1B10 is positively correlated in ACC (Figure 4c). We then measured the expression of HOXA5 in Y1, SW-13 and NCI- H295R cell lines. As shown in Figure 4d and 4e, HOXA5 was also down-regulated in ACC cells lines, especially in NCI-H295R cell line. The bind- ing sequence between HOXA5 and AKR1B10 pro- moter was predicted by JASPAR website (Figure 5a). Next, we overexpressed HOXA5 in NCI- H295R cells (Figure 5b and c) to explore the alteration of AKR1B10 expression. Results showed that AKR1B10 expression was remarkably elevated upon HOXA5 overexpression (Figure 5d and e). Moreover, the interaction between HOXA5 and AKR1B10 was validated by dual-luciferase report
and ChIP assays (figure 5f and g).These results demonstrated that HOXA5 could bind to AKR1B10 and regulate AKR1B10 expression in ACC cells.
Knockdown of HOXA5 partially cancels the effects of AKR1B10 overexpression on NCI-H295R cells proliferation and apoptosis
Finally, to investigate whether HOXA5 could reg- ulate the proliferation and apoptosis of NCI-H295 cells via targeting AKR1B10, we knockdown HOXA5 expression in NCI-H295 cells, and chose the shRNA-HOXA5-1 for the following knock- down experiments owing to its better transfection efficacy (Figure 6a and b). NCI-H295 cells that overexpressed with AKR1B10 were silenced with HOXA5 or not, then the proliferation and apop- tosis of cells were evaluated. As illustrated in Figure 6c, the decreased cell viability caused by AKR1B10 overexpression was significantly enhanced after HOXA5 silence at 72 h post- culture. What’s more, cells overexpressed with AKR1B10 exhibited an obvious fewer colonies compared with normal cells, however, cells that simultaneously silenced HOXA5 exerted markedly increased number of colonies when compared with cells that only overexpressed with AKR1B10 (Figure 6d). The similar results were observed in Figure 6e, which showed that the silence of
a
b
C
6
*
Overall Survival
Disease Free Survival
1.0
5
Low AKR1B10 TPM
1.0
High AKR1B10 TPM
Low AKR1B10 TPM
High AKR1B10 TPM
Logrank p=0.0029
Logrank p=0.021
0.8
HR(high)=0.29
HR(high)=0.45
+
p(HR)=0.0052
0.8
p(HR)=0.024
Percent survival
n(high)=37
n(high)=37
0.6
n(low)=38
Percent survival
0.6
n(low)=38
(
0.4
0.4
2
0.2
0.2
-
0.0
0.0
O
0
50
100
150
0
50
100
150
ACC
Months
Months
(num(T)=77; num(N)=128)
a
b
Relative mRNA expression of AKR1B10
Relative expression of AKR1B10
1.5
1.5
**
1.0
AKR1B10
1.0
0.5
GAPDH
0.5
Y1
SW-13
NCI-H295R
0.0
0.0
Y1
SW-13
NCI-H295R
Y1
SW-13
NCI-H295R
C
d
Relative mRNA expression
Relative expression of AKR1B10
4
2.5
3
2.0
of AKR1B10
AKR1B10
1.5
GAPDH
1.0-
1
Control
Oe-NC
Oe-AKR1B10
0.5
0
Control
Oe-NC
Oe-AKR1B10
0.0
Control
Oe-NC
Oe-AKR1B10
e
Control
f
Control
Oe-NC
Oe-AKR1B10
200
Oe-NC
Oe-AKR1B10
Cell viability (%)
150
100
50
**
0
24 h
48 h
72 h
g
Relative expression of Ki67
1.5
Relative expression of PCNA
1.5.
**
Ki67
1.0
1.0-
PCNA
GAPDH
0.5-
0.5-
Control
Oe-NC
Oe-AKR1B10
0.0
Control
Oe-NC
Oe-AKR1B10
0.0
Control
Oe-NC
Oe-AKR1B10
HOXA5 increased the expression of Ki67 and PCNA in AKR1B10-overexpressing NCI-H295 cells. figure 6f and g revealed that AKR1B10 over- expression markedly increased the number of
apoptotic (Tunel-positive) cells, but HOXA5 knockdown significantly blocked this effect. Consistently, the effect of AKR1B10 overexpres- sion on the expression of proteins related to
a
Control
Oe-NC
Oe-AKR1B10
Tunel
Tunel-positive cells (%)
30
20-
Dapi
10-
0
Merge
Control
Oe-NC
Oe-AKR1B10
b
Relative expression of Bcl-2
1.5
Relative expression of Bax
2.5
**
2.0
1.0-
1.5-
Bcl-2
0.5
1.0-
Bax
0.5
cleaved-caspase3
0.0
Control
Oe-NC
Oe-AKR1B10
0.0
Control
Oe-NC
Oe-AKR1B10
pro-caspase3
cleaved-caspase9
pro-caspase9
Relative expression of cleaved/pro-caspase3
2.0-
2.5
Relative expression of cleaved/pro-caspase9
GAPDH
1.5.
2.0-
1.5-
Control
Oe-NC
Oe-AKR1B10
1.0-
1.0-
0.5
0.5-
0.0
Control
Oe-NC
Oe-AKR1B10
0.0
Control
Oe-NC
Oe-AKR1B10
C
Relative expression of p53
2.5-
Relative expression of p21
2.0
p53
2.0·
1.5
p21
1.5-
GAPDH
1.0-
1.0
Control
Oe-NC
Oe-AKR1B10
0.5-
0.5
0.0
Control
Oe-NC
Oe-AKR1B10
0.0
Control
Oe-NC
Oe-AKR1B10
a
b
C
Overall Survival
HOXA5 vs. AKR1810, 79 samples (ACC)
1.0
Data Source: starBase v3.0 project
Low HOXA5 TPM
5
8
High HOXA5 TPM
Regression (y = 0.4562x - 3.071)
Logrank p=0.043
r = 0.283, p-value = 1.17e-02
0.8
HR(high)=0.44
p(HR)=0.048
AKR1810, Expression level: log2[FPKM+0.01)]
2.5
Percent survival
n(high)=38
6
0.6
n(low)=38
0
0.4
4
-2.5
0.2
2
-5
0.0
..
-7.5
0
50
100
150
-5
-2.5
0
2.5
5
7.5
10
0
Months
HOXA5, Expression level: log2(FPKM+0.01)
(num(T)=77; num(N)=128)
ACC
d
e
Relative mRNA expression
1.5
Relative expression of HOXA5
1.5
**
*
of HOXA5
1.0
HOXA5
1.0
0.5
GAPDH
0.5
Y1
SW-13
NCI-H295R
0.0
0.0
11
SW-13
NCI-H295R
Y1
SW-13
NCI-H295R
apoptosis including Bcl-2, Bax, c-caspase3 and c-caspase9, was also partially canceled upon HOXA5 silence (Figure 6h and i).
Discussion
In the present study, we demonstrated that HOXA5 and AKR1B10 were down-regulated in ACC cells and that the overexpression of AKR1B10 contributed to reduced proliferation and increased apoptosis of ACC cells, but under- expression of HOXA5 was associated with the progression of ACC. In addition, we also demon- strated that HOXA5 could bind to AKR1B10 and enhance its expression in ACC cells. Collectively, we revealed that HOXA5 may exert its anticancer effects through targeting AKR1B10, thereby lead- ing to the inhibition of the abnormal proliferation
and induction of apoptosis via activating p53 sig- naling in ACC cells.
Primary aldosteronism (PA) is one of the com- mon causes of secondary hypertension and can affect up to 13% of hypertensive patients [15]. It has been reported that PA is very likely to cause more serious target organ damage, including car- diovascular and kidney damage, fibrosis, hypertro- phy and vascular inflammation [16-18]. In addition to high risk and high prevalence, PA is also characterized by hyper-secretion of aldoster- one and adrenal hyperplasia, which is due to hyper-aldosterone secreted by adrenal cells and high proliferation of adrenal cells. Most patients with PA have a benign adrenal adenoma or bilat- eral hyperplasia [19,20]. ACC, however, is a very rare cause of increased aldosterone levels. The
Canonical HOXA5 binding motif
L
a
20
ATTA
1.5
8 1.0
TA
0.5
ECC
200
1
2
3
4
$
.
1
.
AKRIB10 promoter
-2000
-1665 ~- 1658
S1
100
GCAATTAG
b
C
Relative mRNA expression
3
Relative expression of HOXA5
**
2.0
1.5-
of HOXA5
2
HOXA5
1.0
1
GAPDH
0.5
Control
Oe-NC
Oe-HOXA5
0
Control
Oe-NC
Oe-HOXA5
0.0
Control
Oe-NC
Oe-HOXA5
0
e
Relative mRNA expression of AKR1B10
Relative expression of AKR1B10
4
2.0
**
3
1.5-
AKR1B10
N
1.0
1
GAPDH
0.5
Control
Oe-NC
Oe-HOXA5
Control
Oe-NC
Oe-HOXA5
0
0.0
Control
Oe-NC
Oe-HOXA5
f
Y
Oe-NC
9
10
M
Relative luciferase activity
Oe-HOXA5
Relative expression of AKR1B10
2.5
8
2.0
6
1.5
4
1.0-
2
0.5-
0
0.0
AKR1B10-WT (S1)
AKR1B10-MUT (S1)
lgG
Anti-HOXA5
a
b
C
Control
Oe-NC
+ Oe-AKR1B10
Relative mRNA expression
1.5-
1.5-
**
200
- Oe-AKR1B10+shRNA-NC
+ Oe-AKR1B10+shRNA-HOXA5
of HOXA5
Relative expression of HOXA5
HOXA5
Cell viability (%)
1.0-
1.0
150-
GAPDH
0.5-
Control
shRNA-NC
0.5
100
0.0
Control
shRNA-NC
shRNA-HOXA5-1
shRNA-HOXA5-2
shRNA-HOXA5-1
shRNA-HOXA5-2
0.0
Control
shRNA-NC
shRNA-HOXA5-1
shRNA-HOXA5-2
50
24 h
48 h
72 h
d
Control
Oe-NC
Oe-AKR1B10
e
Relative expression of Ki67
1.5-
Relative expression of PCNA
1.5.
**
Ki67
1.0
1.0
PCNA
GAPDH
0.5
0.5
Oe-AKR1B10+
Oe-AKR1B10+
Control
Oe-NC
Oe-AKR1B10
Oe-AKR1B10+shRNA-NC
Oe-AKR1B10+shRNA-HOXA5
shRNA-NC
shRNA-HOXA5
0.0
Control
Oe-NC
Oe-AKR1B10
Oe-AKR1B10+shRNA-NC
Oe-AKR1B10+shRNA-HOXA5
0.0
Control
Oe-NC
Oe-AKR1B10
Oe-AKR1B10+shRNA-NC
Oe-AKR1B10+shRNA-HOXA5
f
Control
Oe-NC
Oe-AKR1B10
Oe-AKR1B10+
Oe-AKR1B10+
shRNA-NC
shRNA-HOXA5
Relative expression of Bcl-2
1.5-
2.5-
*
Tunel
Relative expression of Bax
2.0
1.0
**
1.5
0.5
1.0
0.5
Dapi
0.0
Control
Oe-NC
Oe-AKR1B10
Oe-AKR1B10+shRNA-NC
Oe-AKR1B10+shRNA-HOXA5
0.0
Control
Oe-NC
Oe-AKR1B10
Oe-AKR1B10+shRNA-NC
Oe-AKR1B10+shRNA-HOXA5
Merge
g
h
Bcl-2
25-
Bax
4-
Tunel-positive cells (%)
cleaved-caspase3
Relative expression of cleaved/pro-caspase3
4.
Relative expression of cleaved/pro-caspase9
20
3
15-
pro-caspase3
2
10-
cleaved-caspase9
2
5
pro-caspase9
1
1
GAPDH
0
Control
Oe-NC
Oe-AKR1B10
Oe-AKR1B10+shRNA-NC
Oe-AKR1B10+shRNA-HOXA5
Control
Oe-NC
Oe-AKR1B10
Oe-AKR1B10+shRNA-NC
Oe-AKR1B10+shRNA-HOXA5
0
Control
Oe-NC
Oe-AKR1B10
Oe-AKR1B10+shRNA-NC
Oe-AKR1B10+shRNA-HOXA5
0
Control
Oe-NC
Oe-AKR1B10
Oe-AKR1B10+shRNA-NC
Oe-AKR1B10+shRNA-HOXA5
underlying genetic mechanism that leads to ACC and tumor formation is still not fully elucidated.
The data from GEPIA database indicates that the expression of AKR1B10 and HOXA5 is
significantly down-regulated in the tissues of patients with ACC, and is related to the poor survival of patients. Our results showed that AKR1B10 and HOXA5 were also down-regulated
in ACC cell lines, indicating that they may play an important role in the occurrence and progression of ACC. We then overexpressed AKR1B10 in NCI-H295R cells to observe the change of cell proliferation and apoptosis. We found that AKR1B10 overexpression was able to inhibit pro- liferation and promote apoptosis of cells, suggest- ing the inhibitory effect of AKR1B10 on ACC progression. In addition, the p53 signaling was also activated upon AKR1B10 overexpression. p53 and p21 are tumor suppressor proteins that can induce cell cycle arrest and apoptosis of cancer cells [21]. A previous study reported that knock- down of AKR1B10 can significantly inhibit p53- induced apoptosis of CRC cells, while overexpres- sion of AKR1B10 enhances p53-induced apoptosis and inhibits tumor proliferation in vivo [22]. Our data revealed that AKR1B10 may serve as a tumor inhibitor in ACC via activating p53 signaling, thereby inhibiting proliferation and inducing apoptosis of ACC cells.
In CRC, HOXA5 is also down-regulated and its up-expression induces loss of the cancer stem cell phenotype, preventing tumor progression and metastasis [12]. Moreover, HOXA5 has also been reported to cooperate with p53 to play a role in lung cancer mammary tumorigenesis [13,14]. Notably, the data from ENCORI reveals that the expression of HOXA5 and AKR1B10 is positively correlated in ACC, and HOXA5 is predicted to bind to the AKR1B10 promoter after searching JASPAR web- site. Our results demonstrated that HOXA5 over- expression could increase AKR1B10 expression and the interaction between them was confirmed. Therefore, we speculated that HOXA5 may also function as a tumor suppressor in ACC via targeting AKR1B10 and enhancing its expression level. We subsequently knockdown HOXA5 in the presence of AKR1B10 overexpression to investigate whether the effect of AKR1B10 on cell proliferation and apoptosis could be affected. In accordance with our hypothesis, silence of HOXA5 remarkably abol- ished the inhibitory effect of AKR1B10 on cell pro- liferation and the promoting effect of AKR1B10 on cell apoptosis. However, the effect of AKR1B10 on cell proliferation and apoptosis has not been totally reversed by HOXA5 silence. This may be explained by that the expression of HOXA5 has not been fully silenced or maybe there are other proteins and
pathways that regulate AKR1B10, which need to be further elucidated in the following research. Additionally, our research is only performed on in vitro cell model, lacking the validation of in vivo evidence. In the subsequent experiments, we will intend to repeat our experiments in animal models to make our conclusions stronger.
Conclusion
Taken together, our results revealed that HOXA5 could target AKR1B10 to increase AKR1B10 level and activate p53 signaling, thereby inhibiting pro- liferation and inducing apoptosis of ACC cells, ultimately contributing to the suppression of ACC. Our findings suggested that the approaches that targeting HOXA5/AKR1B10 axis may be a promising therapy in treating ACC.
Acknowledgements
Not applicable.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
No funding was received.
Statement of Ethics
Not applicable.
Author contributions
DC and RH contributed to study conception or design; DC, ZS, XC, QW and JZ contributed to data acquisition; DC, FR, YS and HW contributed to analysis or interpretation of data; DC and RH drafted the work and revised it critically for important intellectual content. All authors read and approved the final manuscript.
Highlights
1. AKR1B10 and HOXA5 expression is significantly down- regulated in ACC tissues and cells.
2. AKR1B10 overexpression inhibits proliferation and pro- motes apoptosis of ACC cells.
3. HOXA5 can bind to AKR1B10 promoter to regulate AKR1B10 expression in ACC cells.
4. HOXA5 activates p53 pathway to inhibit ACC progression by binding to AKR1B10.
ORCID
Rongxi Huang @ http://orcid.org/0000-0002-0005-8697
References
[1] Jain M, Zhang L, He M, et al. TOP2A is overexpressed and is a therapeutic target for adrenocortical carcinoma. Endocr Relat Cancer. 2013;20(3):361-370.
[2] Terzolo M, Ardito A, Zaggia B, et al. Management of adjuvant mitotane therapy following resection of adre- nal cancer. Endocrine. 2012;42(3):521-525.
[3] Else T, Kim AC, Sabolch A, et al. Adrenocortical carcinoma. Endocr Rev. 2014;35:282-326.
[4] Rubin B, Pilon C, Pezzani R, et al. The effects of mito- tane and 1alpha,25-dihydroxyvitamin D3 on Wnt/beta — catenin signaling in human adrenocortical carcinoma cells. J Endocrinol Invest. 2020;43(3):357-367.
[5] DiStefano JK, Davis B. Diagnostic and Prognostic Potential of AKR1B10 in Human Hepatocellular Carcinoma. Cancers (Basel). 2019;11(4):112019.
[6] Namba R, Kaneko H, Suzumura A, et al. In vitro epir- etinal membrane model and antibody permeability: rela- tionship with anti-VEGF resistance in diabetic macular edema. Invest Ophthalmol Vis Sci. 2019;60(8):2942-2949.
[7] He YC, Shen Y, Cao Y, et al. Overexpression of AKR1B10 in nasopharyngeal carcinoma as a potential biomarker. Cancer Biomarkers. 2016;16(1):127-135.
[8] Liu W, Song J, Du X, et al. AKR1B10 (Aldo-keto reductase family 1 B10) promotes brain metastasis of lung cancer cells in a multi-organ microfluidic chip model. Acta Biomater. 2019;91:195-208.
[9] Yao Y, Wang X, Zhou D, et al. Loss of AKR1B10 promotes colorectal cancer cells proliferation and migration via regulating FGF1-dependent pathway. Aging (Albany NY). 2020;12(13):13059-13075.
[10] Yan W, Liu S, Xu E, et al. Histone deacetylase inhibi- tors suppress mutant p53 transcription via histone deacetylase 8. Oncogene. 2013;32(5):599-609.
[11] Wang H, Wei H, Wang J, et al. MicroRNA-181d-5p- containing exosomes derived from CAFs promote EMT by regulating CDX2/HOXA5 in breast cancer. molecular therapy. Nucleic acids. 2020;19: 654-667.
[12] Ordóñez-Morán P, Dafflon C, Imajo M, et al. HOXA5 counteracts stem cell traits by inhibiting wnt signaling in colorectal cancer. Cancer Cell. 2015;28(6):815-829.
[13] Gendronneau G, Lemieux M, Morneau M, et al. Influence of Hoxa5 on p53 tumorigenic outcome in mice. Am J Pathol. 2010;176(2):995-1005.
[14] Chang CJ, Chen YL, Hsieh CH, et al. HOXA5 and p53 cooperate to suppress lung cancer cell invasion and serve as good prognostic factors in non-small cell lung cancer. J Cancer. 2017;8(6):1071-1081.
[15] Aji G, Li F, Chen J, et al. Upregulation of PCP4 in human aldosterone-producing adenomas fosters human adrenocortical tumor cell growth via AKT and AMPK pathway. Int J Clin Exp Pathol. 2018;11 (3):1197-1207.
[16] Savard S, Amar L, Plouin PF, et al. Cardiovascular complications associated with primary aldosteronism: a controlled cross-sectional study. Hypertension. 2013;62(2):331-336.
[17] Mulatero P, Monticone S, Bertello C, et al. Long-term cardio- and cerebrovascular events in patients with primary aldosteronism. J Clin Endocrinol Metab. 2013;98(12):4826-4833.
[18] Chou CH, Chen YH, Hung CS, et al. Aldosterone impairs vascular smooth muscle function: from clinical to bench research. J Clin Endocrinol Metab. 2015;100 (11):4339-4347.
[19] Zennaro MC, Fernandes-Rosa F, Boulkroun S, et al. Bilateral idiopathic adrenal hyperplasia: genetics and beyond. Horm Metab Res. 2015;47(13):947-952.
[20] Jouinot A, Armignacco R, Assie G. Genomics of benign adrenocortical tumors. J Steroid Biochem Mol Biol. 2019;193:105414.
[21] Kim EM, Jung CH, Kim J, et al. The p53/p21 complex regulates cancer cell invasion and apoptosis by target- ing Bcl-2 family proteins. Cancer Res. 2017;77 (11):3092-3100.
[22] Ohashi T, Idogawa M, Sasaki Y, et al. AKR1B10, a transcriptional target of p53, is downregulated in colorectal cancers associated with poor prognosis. Mol Cancer Res. 2013;11(12):1554-1563.