Expression of clock-related genes in benign and malignant adrenal tumors
Anna Angelousi1 . Narjes Nasiri-Ansari2 . Angeliki Karapanagioti2 . Georgios Kyriakopoulos2 . Chrysanthi Aggeli3 . Giorgos Zografos3 . Theodosia Choreftaki4 . Christos Parianos3 . Theodora Kounadi5 . Krystallenia Alexandraki6 . Harpal S. Randeva7 . Gregory Kaltsas6 · Athanasios G. Papavassiliou2 · Eva Kassi1,2
Received: 8 November 2019 / Accepted: 26 February 2020 @ Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Although the effect of the central clock system on adrenal function has been extensively studied, the role of the peripheral clock system in adrenal tumorigenesis remains largely unexplored. In this study we investigated the expression of clock- related genes in normal adrenocortical tissue and adrenocortical tumors. Twenty-seven fresh frozen human adrenal tissues including 13 cortisol secreting adenomas (CSA), seven aldosterone producing adenomas (APA), and seven adrenocortical carcinomas (ACC) were collected. CLOCK, BMALI, PER1, CRY1, Rev-ERB, and RORa mRNA and protein expression were determined by qPCR and immunoblotting in pathological tissues and compared with the adjacent normal adrenal tissues. A significant downregulation of PER1, CRY1, and Rev-ERB compared with their normal tissue was demonstrated in CSA. All clock-related genes were overexpressed in APA compared with their normal tissue, albeit not significantly. A significant upregulation of CRY1 and PERI and downregulation of BMALI, RORa, and Rev-ERB compared with normal adrenal tissue was observed in ACC. BMAL1 and PER1 were significantly downregulated in APA compared with CSA. CLOCK, CRY1, and PERI were upregulated, whereas BMALI, RORa, and Rev-ERB were downregulated in ACC compared with CSA. Our study demonstrated the expression of CLOCK, BMALI, PER1, CRY1, Rev-ERB, and RORa in normal and pathological human adrenal tissues. Adrenal tumors exhibited altered expression of these genes compared with normal tissue, with specific differences between benign and malignant lesions and between benign tumors arising from glomerulosa vs fasciculata zone. Further studies should clarify whether these alterations could be implicated in adrenocortical tumorigenesis.
Keywords Adrenal . Clock genes . Cortisol secreting adenoma . Aldosterone producing adenoma . Adrenocortical carcinoma
These authors contributed equally: Anna Angelous, Narjes Nasiri- Ansari
☒ Eva Kassi evakassis@gmail.com
1 1st Department of Internal Medicine, Laiko University Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece
2 Department of Biological Chemistry, Medical School, National and Kapodistrian University of Athens, Mikras Asias 75, Goudi, 11527 Athens, Greece
3 3rd Department of Surgery, General Hospital of Athens “G. Gennimatas”, Athens, Greece
Introduction
The hypothalamicuitary-adrenal (HPA) axis is an ideal model for studying the transmission of circadian informa- tion in the body. Adrenal gland is still of prime importance
4 Department of Pathology, General Hospital of Athens “G. Gennimatas”, Athens, Greece
5 Department of Endocrinology and Diabetes Center, Athens General Hospital “G. Gennimatas”, Athens, Greece
6 1st Department of Propaedeutic Internal Medicine, Laiko University Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece
7 Division of Translational and Experimental Medicine, Metabolic and Vascular Health, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK
for understanding how the oscillations of clock genes in peripheral tissues result. However, most of our knowledge of the peripheral clock regulation comes from in vitro and animal studies, whereas data derived from human tissues are scarce [1, 2].
It has been more than 30 years since adrenal gland was shown to exhibit an intrinsic rhythmicity of corticosterone secretion in mice [3, 4]. Recently, whole-genome micro- array hybridization performed to characterize the circadian transcriptome of the murine adrenal gland showed that ~5% of the murine adrenal genome is under circadian control [5].
In general, the central clock in the suprachiasmatic nucleus (SCN) could entrain the peripheral oscillators in the adrenal gland via the three pathways: (a) the humoral pathway of the HPA-axis, (b) the neural pathway of the autonomic nervous system, and (c) a local adrenocorti- cotropic hormone (ACTH) secretion [6]. Indeed, adrenal denervation led to an abolishment of both the circadian corticosterone rhythm, as well as of the daily variation of the adrenal responsiveness to ACTH [7-9]. SCN and adrenal gland maintain their intrinsic rhythm via a transcriptional/translational feedback loop that consists of positive and negative regulators in a 24-h time-of-day pattern. The core loop is formed by the CLOCK- BMAL1 heterodimer that stimulates the transcription of period (PER1, PER2, PER3), cryptochrome1, and cryptochrome2. PERs and CRYs proteins heterodimerise and inhibit the transcription of the CLOCK-BMALI. Consequently, CLOCK-BMAL1 activity drops, which reduces the transcription of PER and CRY genes, thereby activating CLOCK/BMAL1 again. Furthermore, Rev- ERBs and RORs can bind to the BMAL1 promoter, forming an additional interlocking transcriptional loop that exhibits auxiliary function to the BMAL1 expression [10, 11].
Recent clinical and in vitro studies have shown that the disruption of central and/or peripheral clock system in var- ious tumors such as breast, lung, and prostate cancer, as well as in hematological malignancies involved in tumourigen- esis, through several pathways [12]. However, data on adrenal tumors are scarce. Moreover, despite the knowledge gained from studies on animal models, the significance of clock-related genes in human adrenal function remains lar- gely unexplored.
Therefore, in the present study we investigated for the first time the mRNA and protein expression of six clock- related genes CLOCK, BMALI, CRY1, PER1, RORa, and Rev-ERB in benign and malignant human adrenocortical tumors and compared them with adjacent normal adrenal tissues.
Methods
Subjects and methods
Subjects
All fresh frozen adrenal tissues were collected prospectively from November 2016 to December 2017 from patients undergoing laparoscopic adrenalectomy either because of tumor size or autonomous cortisol or aldosterone secretion. All surgical procedures were performed in the morning (between 7.00 and 10.00 a.m.) by the same surgical team of the 3rd Department of Surgery in the General hospital “G. Gennimatas” in Athens. For every sample of the patholo- gical adrenal tissue, the surgeon collected also a sample of the adjacent adrenal tissue, which was considered macro- scopically normal, except for adrenocortical carcinomas (ACC) tissues. The adjacent adrenal tissue was used as “control” only when it was histologically confirmed by the pathologist as normal adrenal tissue. The mRNA and pro- tein levels of clock-related genes in ACC tissues were compared with adjacent normal tissues of cortisol secreting adenomas (CSA).
The diagnosis of the autonomous hormonal secretion from the adrenal tumor was based on the history, clinical examination, and preoperative endocrine tests performed in the same laboratory. Cortisol (basal and after 1-mg over- night dexamethasone test (ODST)) and 24-h urinary free cortisol (UFC) levels were measured by electro- chemiluminescent bridging immunoassay (ECLIA) (Cobas 8000 e801, Hitachi, intra-assay CV <3.9% and inter-assay CV <3.8% for all hormones), and ACTH was measured by chemiluminescent assay (Liaison, DiaSorin, intra-assay CV 4.3-7.5% and inter-assay 10-14.5%). Aldosterone and plasma renin activity (PRA) levels were measured by radioimmunoassay.
The histopathological analysis of the surgical specimens was performed by two independent pathologists in the Department of Pathology of “G. Gennimatas” and “Evan- gelismos” General Hospitals in Athens. All patients gave their written informed consent for the use of samples and clinical data, and the protocol of the study was approved by our local ethics committee (Laiko Hospital, code 474) according to the guidelines of the Declaration of Helsinki.
Materials (tissues collection and storage)
A quantity of 100-200 mg of adrenal tissue (adrenal tumor and normal adrenal tissue) was cut and stored immediately after surgery at -80 ℃. A 3 mm slide was cut and used for our analysis within less than 2 months since storage.
Total RNA isolation and qPCR
Total RNA was isolated from 50 mg of adrenal lesion and normal tissue using NucleoSpin® RNA Plus kit (MACHEREY-NAGEL). One microgram of RNA was reverse transcribed to cDNA using a LunaScript RT SuperMix Kit (New England Biolabs). The mRNA expression of CLOCK, BMALI, CRY1, PER1, RORa, and Rev-ERB was evaluated by quantitative real-time poly- merase chain reaction (qRT-PCR) as previously described [13]. Briefly, SYBR Green-based reactions were conducted on the BIORAD CFX96 Touch RT-PCR. All reactions were carried out in triplicates. Each qPCR run included a nega- tive control as well as cDNA from MCF-7 cells, which was used as an internal control to correct the inter-assay varia- tion for samples run on different plates. The relative fold change was calculated using the 2(-Delta Delta C(T)) (AACTs) [14]. Primer sequences for the genes CLOCK, BMAL1, PER1, CRY1, RORa, Rev-ERB, and housekeeping B-actin were used as described previously [13].
Protein extraction and western blot analysis
Protein was extracted from 50 mg adrenal tissues using 2X cell lysis buffer (CST. 9803) according to the manufacturer’s instruction, the protein concentration was determined by Bradford assay (Applichem), and the samples were stored at -80℃ until used. Fifty micrograms of each total protein extract were loaded into each well across the SDS-PAGE gel resolved by electrophoresis. Proteins separated by SDS- PAGE were immediately transferred to nitrocellulose mem- brane. The blotting membrane was blocked with 5% skim milk in 1X-PBST for 1 h at RT. After blocking, blots were incubated with primary antibodies against CLOCK (sc- 271603, Santa Cruz Biotechnology), BMAL1 (sc-365645, Santa Cruz Biotechnology), PER1 (sc-398890, Santa Cruz Biotechnology), CRY1 (sc-393466, Santa Cruz Biotechnol- ogy), and ß-actin (MAB1501, Millipore) overnight at 4 ℃. Blots were washed with 0.1% tween 20 in PBS and incubated with HRP conjugated-secondary anti-mouse (31430, Thermo Scientific) antibody for 1 h at room temperature. The protein bands were visualized using the Clarity Western ECL Sub- strate (Bio-Rad) and quantified using ImageJ software. ß-actin served as a loading control and MCF-7 total protein extract was loaded in each gel to correct the inter-assay variation.
Statistical analysis
All the data are reported as mean ± standard error (STDEV) and median levels. Nonparametric values were analyzed with Mann-Whitney test for the comparison between patients and controls, while Wilcoxon test was performed for the comparison of nonparametric paired values in the
same group. Spearmen rank correlation coefficient test was used for the correlation analysis. All statistical analysis were performed using GraphPad Prism 7 Software. Differences were considered significant at p<0.05.
Results
Characteristics of the studied population
The study included a total of 27 patients with unilateral adrenal tumors, 13 males and 14 females, with a mean age of 54 + 15.7 years old. All patients underwent unilateral adrenalectomy. Histopathological analysis confirmed the diagnosis of 20 benign tumors (adenomas) (13 CSA and seven aldosterone producing adenomas (APA)) and seven ACC (three had both cortisol and androgen hypersecretion and four were nonfunctional). Clinical, biochemical, and histological data are presented in Table 1.
The expression of CLOCK, BMAL1, CRY1, PER1, Rev- ERB, and RORa in benign adrenal tumors
The expression of mRNA (AACTs) and protein (four pro- teins-western blot) of all six clock-related genes was increased in APA compared with their paired adjacent normal tissues, although this upregulation did not reach statistical significance as shown in Fig. 1a, b. On the con- trary, the mRNA expression (AACTs) of all the six clock- related genes (except for RORa) was downregulated in CSA compared with their paired adjacent normal adrenal tissues as it shown in Fig. 2. However, only CRY1, Rev-ERB, and PER1 mRNAs were significantly reduced in CSA compared with their paired adjacent normal tissues (p = 0.03 and p = 0.02, p = 0.05, respectively), whereas no significant chan- ges were observed in CLOCK, BMAL1, and RORa as shown in Fig. 2. At protein level, no significant differences were observed in CSA compared with their paired adjacent nor- mal adrenal tissues a shown in Fig. 4.
Moreover, comparing the AACTs ratio of adenomas/ paired adjacent normal tissue of APA vs CSA, all six clock- related genes showed decreased expression in APA compared with CSA, while only PER1 and BMAL1 were significantly downregulated (p=0.04 and p=0.03, respectively) and CLOCK, CRY1, RORa, and Rev-ERB mRNAs showed no significant changes as it is shown in Fig. 3.
The expression of CLOCK, BMAL1, CRY1, PER1, Rev- ERB, and RORa in adrenocortical carcinomas (ACC)
The mRNA (AACTs) expression of the six clock-related genes showed a different pattern in the ACC tissues. Specifically, CRY1 and PER1 mRNA were significantly upregulated in
| Characteristics of the studied population | Cortisol secreting adenomas | Conn adenomas | ACC |
|---|---|---|---|
| Number | 13 | 7 | 7 |
| F/M | 9/4 | 3/4 | 2/5 |
| Age (mean ± SD, median), years | 52.6± 16 (56) | 51 ± 15 (59) | 65.5±12 (65) |
| Size (cm) (mean ± SD, median) | 5.5 ±2.08 (5.25) | 2.9 ±1.8 (2) | 7.6±2.5 (7.2) |
| ODST (ug/dl) (mean ± SD, median, normal <1.8) | 11.8 ±8.4 (6.7) | nd | 17.5 ±2 (17) |
| F 8:00 a.m. (ug/dl) (mean ± SD, median, normal =7-18) | 22.6 ±7.9 (21) | 16±4(18.6) | 24 ±2.5 (25) |
| ACTH (pmol/l) (mean ± SD, median, normal=9-37) | 7.5±3.8 | nd | <3 |
| UFC (µg//24 h) (mean ± SD, median, normal =10-50) | 154 ±110 (125) | 54 ±25 (42) | 175.5±100(175) |
| Aldosterone (ng/dl) (mean ± SD, median, normal =2-15) | 11.2±2.2 (12) | 35.2 ± 18.6 (25.5) | nd |
| PRA (ng/ml/h) (mean ± SD, median, normal =1.9-3.7) | 2±0.3 (1.9) | 0.9 ±0.4 (0.64) | nd |
| Ki-67% (mean ± SD, median) | nd | nd | 22.5 ±8 (22.5) |
ACC adrenocortical carcinoma, F/M female/male, SD standard deviation, ODST overnight dexamethasone suppression test, F cortisol, ACTH adrenocorticotropic hormone, UFC urinary free cortisol, PRA plasma renin activity
a
CLOCK
BMAL-1
b
4-
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mRNA (AACt) levels|
2-
mRNA(AACT) levels
6-
I.C.
P1.P
P1.A
P2.P
P2.A
P3.P
P3.A
P4.P
P4.A
4-
CLOCK
0-
2-
-2-
0-
BMAL-1
-4-
.
-2-
-6
P=0.37
-4
P=0.4
CRY1
Adjacent Normal
APA
Adjacent Normal
APA
PER1
10-
PER1
10-
CRY1
mRNA(AACT) levels
mRNA(AACT) levels
5-
8-
Actin
6-
0
·
4-
CLOCK
BMAL-1
0,3
1
-5-
2-
Protein levels
Protein levels
P=0.25
P=0.6
0,2
0,8
-10
0,6
Adjacent Normal
APA
0
Adjacent Normal
APA
0,1
0,4
0,2
0-
RORa
4-
Rev-Erb
0
0
mRNA(AACT) levels
mRNA(AACT) levels
Control
APA
Control
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2-
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0-
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3
5
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Protein Levels
Protein levels
4
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3
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Adjacent Normal
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-6
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1
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1
0
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Control
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ACC compared with normal adrenal tissues (p=0.02 and p = 0.04, respectively), whereas BMAL1, RORa, and Rev- ERB mRNAs were significantly downregulated (p= 0.0048, p=0.007 and 0.028, respectively) as shown in Fig. 2.
Comparing ACC with CSA, we found that CLOCK, CRY1, and PER1 mRNA were significantly upregulated (p=0.017, p = 0.003, and p = 0.001, respectively), whereas BMAL1, Rev-
ERB, and RORa were significantly downregulated (p = 0.0097, p = 0.036, and p = 0.006, respectively) as shown in Fig. 2. At protein level the expression of CLOCK and CRY1 was mar- ginally increased in ACC compared with CSA reaching sta- tistical significance (p=0.09 and p = 0.08, respectively). No significant difference was found between CSA and ACC and/ or between ACC and normal adrenal tissues in BMAL1 and PER1 protein expression as shown in Fig. 4.
CLOCK
BMAL-1
PER1
10
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20
20
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30
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40
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CSA
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CLOCK
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mRNA Levels (AACT A/ AACT N)
mRNA Levels (AACTA/ AACT N)
PER1 *
5-
80
mRNA Levels (AACT A/ AACT N)
10-
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0-
60-
40-
5-
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20-
0
0-
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P=0.04
P=0.035
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mRNA Levels (ΔΔΣΤ Α/ ΔΔΣΤ Ν)
15.
CRY1
mRNA Levels (AACT A/ AACT N)
200-
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mRNA Levels (AACT A/ AACT N)
20-
Rev-Erb
10-
150-
15-
100-
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5
50-
5-
0
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APA
Correlation between clock genes expression and adrenal tumors characteristics
No significant correlation was found between clock gene expression and the size of tumors (CSA, APA, and ACC),
UFC (CSA), aldosterone levels, and PRA (APA) as well as Ki-67% in ACC. However, post 1-mg ODST, cortisol level was positively correlated with Rev-ERB (r = 1.0, p = 0.02) and negatively correlated with CLOCK expression (r= -0.14, p =0.001) in CSA as it is shown in Table 2.
a
b
Cortisol Secreting Adenomas
Adrenocortical Carcinoma
I.C. P1.P P1.A P2.P P2.A P3.P P3.A P4.P P4.A
I.C.
P1
P2
P3
P4
P5
P6
P7
CLOCK
CLOCK
BMAL-1
BMAL-1
CRY1
CRY1
PER1
PER1
a-Actin
a-Actin
c
CLOCK
BMAL-1
CRY1
2
15
2
Protein levels
1,5
Protein levels
Protein levels
10
1,5
1
1
0,5
5
0,5
0
Adjacent Normal
P=0.09
0
CSA
ACC
0
P=0.08
Adjacent Normal
CSA
ACC
Adjacent Normal
CSA
ACC
PER1
2
Protein levels
1,5
1
0,5
0
Adjacent Normal
CSA
ACC
| Genes/r, p | CSA/APA/ACC Size (cm) | CSA ODST (µg/dl) | CSA UFC (ug/24 h) | APA Aldosterone | ACC Ki-67% |
|---|---|---|---|---|---|
| BMAL1 | 0.16, p= 0.7/0.6, p = 0.5/-0.3, p = 0.6 | -0.2, p=0.8 | -0.50, p=0.3 | -0.5 (p=0.9) | 0.4, p = 0.66 |
| CLOCK | 0.42, p=0.3/1, p=0.33/-0.2, p=0.8 | -0.14, p<0.001 | -0.5, p=0.2 | -1.0 (p=0.3) | 0.9, p=0.3 |
| RORa | -0.8, p=0.1/1, p=0.3/0.2, p=0.8 | 0.9, p = 0.08 | 0.08, p = 0.9 | -0.5 (p=0.9) | -0.4, p=0.6 |
| Rev-ERB | -0.54, p = 0.2/not possibleª/0.3, p = 0.6 | 1.0, p = 0.02 | -0.08, p =0.9 | 0.5 (p=0.9) | -0.4, p=0.66 |
| CRY1 | 0.02, p = 0.9/-0.5, p = 0.9/0.3, p = 0.6 | 0.7, p=0.2 | -0.5, p=0.3 | 1.0 (p=0.3) | 0, p= 1 |
| PER1 | -0.2, p=0.6/-0.5, p=0.9/0.6, p =0.5 | 0.8, p = 0.1 | -0.5, p=0.3 | 0.5 (p=0.3) | -0.9, p=0.3 |
ODST Overnight dexamethasone suppression test, UFC Urinary free cortisol
ªDue to small sample
Discussion
Our results revealed the existence of a local circadian clock system in the human adrenal cortex showing evidence of mRNA and protein expression of the six clock-related genes CLOCK, BMALI, CRY1, PER1, RORa, and Rev-
ERB. The expression of these genes seems however to be dysregulated in adrenal tumors exhibiting inappropriate alterations of the PER1, CRY1, RORa, and Rev-ERB expression according to BMAL1/CLOCK changes, sug- gesting thus a disruption of the known feedback loops. Furthermore, circadian clock dysregulation appears to
differ between tumors originating from the glomerulosa and the fasciculata zone as well as between benign (CSA) and malignant adrenal tumors (ACC). Concerning clock genes expression and tumor functionality, Rev-ERB expression was positively correlated with the degree of autonomy of CSA (less suppression of cortisol secretion in the ODST), whereas CLOCK expression was inversely correlated with the degree of autonomy of CSA.
Recent data from in vitro and animal studies have clearly demonstrated the circadian expression of clock genes in the adrenal cortex as well as in the adrenal medulla [3-6]. In adrenal cortex and medulla of mice, PER1, CRY2, and BMAL1 protein expression displayed day/night variation with PER1 and CRY2 levels peaking in the middle of the light phase, whereas BMAL1 in the dark phase [5, 6, 15]. Adrenal explants from capuchin monkeys found to express BMAL1 and PER2 mRNA in oscillatory fashion (higher expression of BMAL1 during the night and PER2 during the morning) while a circadian rhythm of StAR expression and corticosterone secretion was also demonstrated [16]. Similarly, in hypophysectomised animals the adrenal gland was still able to maintain the corticosteroid circadian rhythmicity [1, 17, 18] suggesting that the adrenal oscil- lator can act independently of humoral SCN signaling via the pituitary gland [19]. Another study revealed a translo- cation of PER1 protein from the cytoplasm to the nucleus during the daily cycle, supporting the existence of a core oscillator in the individual adrenal gland cells [6, 19-21]. In our study we did not evaluate the circadian expression of the clock genes in the adrenal tissues since all surgical samples were collected during surgical procedures between 07:00 and 10:00 a.m.
A labeling of PER1, PER2, and BMAL1 mRNA in the inner zonas of rat adrenal tissues has also been observed by in situ hybridization, which was particularly strong in the reticularis zona [19]. In line, PER1 protein was visualized in the cells of the rat adrenal cortex with a higher intensity of immunostaining in the inner zone. These data along with our findings strengthen the hypothesis of the existence of differences in the expression of the circadian clock genes between the different zones of the adrenal cortex.
Although clock genes expression has been extensively reported in the nonhuman primate and rodent adrenal gland, data in human are scarce. To the best of our knowledge, there is only one study reporting the expres- sion of PER1, PER2, CRY2, CLOCK, and BMAL1 mRNA in human normal adrenal tissues, with a PER1 mRNA predominance, whereas the remaining genes were only weakly expressed as it was evaluated by conventional PCR [2]. In the present study, we demonstrate mRNA and protein expression of CLOCK, BMAL1, PER1, CRY1, as well as-for the first time-of RORa and Rev-ERB in the human adrenal cortex.
There are also data indicating that human adrenocortical cells (H295R) present a rhythmic expression of PER1, PER2, CRY1, and BMAL1 modulated by the administration of glucocorticoids [22]. These alterations occurred inde- pendently from ACTH and corticotropin-releasing hormone [22]. Moreover, downregulation of PER1 and/or pharma- cological blockade of PER1 nuclear entry in the H295R cell line by casein kinase 1-8/8 inhibitor PF-670462 [23] was associated with lower plasma aldosterone levels and reduced 3ß-hydroxysteroid dehydrogenase (HSD3B) expression in this cell line [23], while PER1 increased the expression of both 11ß-hydroxylase and aldosterone syn- thase reporter genes in H295R cells [24]. Accordingly, we found increased expression of PER1 in APA. Moreover, a recent study showed that CRY1 mRNA was upregulated in human APA compared with the paired adjacent normal adrenal cortical tissue [25], while treatment of human adrenocortical cells (HAC15 cells) with angiotensin II resulted in a significant upregulation of CRY1 and down- regulation of CRY2 through activation of the angiotensin type 1 receptor [25].
There are studies supporting that steroid hormones can regulate the adrenal clock genes expression and vice versa [16, 23-27]. Since the adrenal tumor and the adjacent normal adrenal tissue, which are both exposed to the same glucocorticoid millieu, showed different clock gene expression, one could speculate that the initial step of the tumorigenesis is the perturbation of the local circadian clock system. However, there are processes such as acetylation or methylation of glucocorticoid receptor that could be pri- marily disturbed and locally-within the tumor-alter the sensitivity to glucocorticoids leading to a divergent circa- dian clock genes profile. Moreover, there are other factors such as melatonin, sirtuins, and casein kinase 1 that could primarily be disturbed and could also implicated in the local -within the tumor-perturbation of the clock genes expression (Fig. 5).
All these data suggest a complex and multifactorial mechanism involved in the dysregulation of cortisol secre- tion from the adrenal cortex, which may include apart from the known ACTH stimuli or peripheral adrenal innervation a number of known, or still unknown factors influencing the cortisol production either directly or through circadian clock genes disruption. Based on the type of our study, we should better avoid favouring one scenario over the other; thus the hormonal dysregulation could either precede the altered expression of circadian clock genes or follow it.
Interestingly, several studies have shown that CLOCK system can exert important influence on the susceptibility to phenotypes of psychiatric and metabolic disorders as well as appetite disturbances [28-30]; these data were based on analysis of clock genes’ expression in tissues mainly involved in these disorders, such as hypothalamic and
CRH
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SIRT-1 Enhance GC transcriptional activity
Down-regulation of nGRE- containing genes
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GR
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Down regulation of GRE- containing genes such as Per, Cry, ..
Rev-ERB
ROR
SIRT-1
CK1
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adipose tissues or in blood cells. It is well known that Cushing syndrome is characterized by psychiatric, eating, and metabolic disorders. Herein, we identified an altered clock genes expression in adrenal tissues of Cushing patients, however it would be somehow risky to extrapolate our results from adrenal tissues to clinical phenotypes.
Concerning clock genes expression in ACCs we found that CLOCK, CRY1, and PER1 were upregulated com- pared with the normal tissue, whereas BMAL1 and RORa were downregulated. This is in line to a previous study showing higher expression of CLOCK in human colorectal cancer compared with healthy tissue [31]. In contrast to our findings, BMAL1 was found to be upregulated in cer- tain types of pleural mesothelioma; subsequent experi- ments revealed reduced cell growth and induced apoptosis upon BMAL1 knockdown in tumorigenic cells, but not in
maintenance of circadian rhythm in physiological conditions. CK-1 phosphorylate the Per-Cry complex, whereas Sirt-1 induces GC-GR transcriptional activity, negatively regulates CLOCK-BMAL1 activ- ity, and finally promote the degradation of the PER protein. CRH corticotropin-releasing hormone; ACTH adrenocorticotropic hormone; GR glucocorticoid receptor; GRE glucocorticoid response element; nGRE negative GRE; CK-1 casein kinase 1; Sirt-1 sirtuin-1; A acetylated GR
cells derived from healthy tissue [32]. Thus, it could be speculated that epigenetic alterations of various cancer cell types could be responsible for the modulating influence of the circadian clock on proliferation, apoptosis, and cell cycle progression [33].
Indeed, in vitro and in vivo studies have shown that clock genes may influence cancer susceptibility by regulating nucleotide excision repair, DNA damage checkpoints or even apoptosis [34, 35], whereas by resetting the circadian clock in tumor cells a slow down or even inhibition of tumor growth was achieved [36]. Kiessling et al. showed that treatment of B16 mouse melanoma cells with dexamethasone, forskolin, or heat shock protein restored rhythmic clock gene expres- sion and significantly slowed down B16 cell proliferation. The above findings support the notion that peripheral clock system could be a potential therapeutic target.
One limitation of this study is the small number of tissues samples. Moreover, since it is a cross-sectional study, we cannot draw conclusions regarding causal relation between dysregulation of peripheral clock system and adrenal tumorigenesis and our results should be interpreted with caution. In our study the correlation of CLOCK and Rev- ERB with cortisol levels following the ODST and with the degree of adrenal autonomy may also imply a possible role of the clock genes in the steroidogenesis. However, further in vitro and in vivo functional studies are needed to confirm these findings. Evaluation of the diurnal expression of clock-related genes with multiple consecutive time-points in normal adrenal tissue and adrenal tumors could shed light in the role of the peripheral clock system in the adrenal tumorigenesis, however, this kind of study is difficult to be conducted in human.
Conclusions
Herein, we demonstrate the expression of CLOCK, BMAL1, CRY1, PER1, RORa, and Rev-ERB in normal human adrenal cortex as well as in benign and malignant human adrenocortical tumors. Moreover, we report a dysregulation of the peripheral clock system with different pattern of clock-related genes expression in benign vs malignant tumors as well as in tumors originating from the glumerulosa vs fasciculata zona. The latter may suggest a different interplay between HPA axis as well as renin-angiotensin-aldosterone axis and peripheral clock system. Understanding the molecular basis of peripheral circadian oscillators in adrenal tissue is essential to develop treatments against clock-related disorders such as adrenal tumors. At last studying the correlation of clock genes alterations with the alterations in the expression of other genes known to have a causative role in adrenal tumorigenesis such as b-catenin, protein kinase (PRKACA, PRKR1A), or cell cycle regulation genes can lead to a better understanding of the pathophysiology of adrenal tumors.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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