ORIGINAL ARTICLE
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What is the role of CHCHD2 in adrenal tumourigenesis?
Angeliki Karapanagioti1,2 . Narjes Nasiri-Ansari1 . Athanasios Moustogiannis3 . George C. Trigas4 .
Georgios Zografos5 . Chrysanthi Aggeli5 . Georgios Kyriakopoulos6 . Theodosia Choreftaki7 . Anastassios Philippou3 . Gregory Kaltsas2 . Eva Kassi1,2 . Anna Angelousi OD8
Received: 21 December 2022 / Accepted: 1 May 2023 / Published online: 23 May 2023 @ The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2023
Abstract
Purpose CHCHD2 is an antiapoptotic mitochondrial protein acting through the BCL2/BAX pathway in various cancers. However, data on the regulatory role of CHCHD2 in adrenal tumourigenesis are scarce.
Methods We studied the expression of CHCHD2, BCL2, and BAX in human adrenocortical tissues and SW13 cells. mRNA and protein levels were analyzed through qPCR and immunoblotting, respectively, in 16 benign adrenocortical neoplasms (BANs), along with their adjacent normal adrenal tissues (controls), and 10 adrenocortical carcinomas (ACCs). BCL2/BAX mRNA expression was also analyzed in SW13 cells after CHCHD2 silencing. MTS, flow cytometry and scratch assays were performed to assess cell viability, apoptosis, and invasion, respectively.
Results BCL2 and CHCHCD2 mRNA and protein expression was increased in BANs compared to normal adrenal tissues whereas BAX was decreased. BAX and CHCHD2 mRNA and protein levels were significantly downregulated and upre- gulated, respectively, in ACCs compared with either BANs or controls. Expression of the studied genes was not different among cortisol-secreting and nonfunctional ACAs. No significant association was found between genes’ expression and other established prognostic markers of ACCs patients. In vitro analysis showed that CHCHD2 silencing resulted in reduced cell viability and invasion as well as increased SW13 cells apoptosis.
Conclusions CHCHD2 expression seems to be implicated in adrenal tumourigenesis and its absence resulted to increased apoptosis in vitro. However, the exact mechanism of action and particularly its association with the BAX/BCL2 pathway needs to be further studied and evaluate whether it could be a protentional therapeutic target.
Keywords CHCHD2 . BAX . BCL2 . Apoptosis . Adrenocortical carcinoma . Adrenal adenomas
These authors contributed equally: Angeliki Karapanagioti, Narjes Nasiri-Ansari, Athanasios Moustogianni
☒ Anna Angelousi a.angelousi@gmail.com
1 Department of Biological Chemistry, Medical School, National and Kapodistrian University of Athens, Athens, Greece
2 1st Department of Propaedeutic Internal Medicine, Laikon University Hospital, National and Kapodistrian University of Athens, Athens, Greece
3 Department of Experimental Physiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece
4 Department of Histology and Embryology, Medical School, National and Kapodistrian University of Athens, Athens, Greece
Introduction
Adrenocortical carcinoma (ACC) is a highly aggressive malignancy with an estimated worldwide prevalence of 0.7-2.0 cases per million adults and a five-year-survival ranging from 16 to 80% depending on local invasion and
5 3rd Department of Surgery, General Hospital of Athens “G. Gennimatas”, Athens, Greece
6 Department of Pathology, Evangelismos General Hospital, Athens, Greece
7 Department of Pathology, General Hospital of Athens “G. Gennimatas”, Athens, Greece
8 1st Department of Internal Medicine, Laikon University Hospital, National and Kapodistrian University of Athens, Athens, Greece
the metastatic status [1, 2]. The number of mitoses, the Ki- 67 labeling index value and a Weiss score of more than 3 are the most utilized tools in distinguishing benign (BAN) from malignant adrenocortical carcinomas (ACC) and constitute predictive markers of poor prognosis for ACC in everyday practice [3, 4]. The last decade there has been an enormous progress in the understanding of the molecular biology of adrenocortical neoplasms through transcriptome analysis in order to provide potential prognostic markers [5] that are though not widely available in clinical practice [6].
The management of patients with ACC requires a mul- tidisciplinary approach. Complete surgical resection is the treatment of choice, whereas mitotane is the only currently available adrenolytic medication achieving an overall response of approximately 30%. In metastatic or locally advanced disease the combinations of etoposide, doxor- ubicin, and cisplatin with mitotane (EDP-M) has been shown to achieve a longer median progression-free survival (PFS) of 5.0 months compared to 2.1 months of strepto- zotocin based schemes. However, this effect is associated with impairment of the quality of life in these patients due to the toxicity and the serious adverse events of this treat- ment without exhibiting a statistical significant difference in overall survival (OS) [2].
The overall survival rate of patients with advanced ACC still remains low since ACC is resistant to the available systemic or local therapies including chemotherapy and radiation therapy. There is a need to explore new ther- apeutic approaches targeting molecular pathways con- sidered to be involved in development and progression of ACC including those of autophagy and apoptosis that could also induce resistance to chemotherapy [7, 8].
Mitochondrial outer membrane permeabilization (MOMP) is a critical control point in apoptosis resulting in the release of pro-apoptotic mitochondrial contents. Recently it has been shown that MOMP is largely con- trolled by the BCL2 family proteins such as BAX, which under apoptotic stress becomes activated and oligomerizes on the outer mitochondrial membrane. This leads to dif- fusion of the mitochondrial contents into the cytoplasm and subsequent activation of the caspase cascade. BAX is regulated primarily by anti-apoptotic BCL2 proteins including BCL-XL, which prevents BAX from accumu- lating into the mitochondria [8]. In response to apoptotic stimuli, Coiled-coil helix domain-containing 2 (CHCHD2) protein encoded by the CHCHD2 gene located on human chromosome 7p11.2, decreases and loses its mitochondria localization, accompanied by decreased BCL-BAX interaction and increased BAX homo-oligomerization [7-9]. Subsequently, CHCHD2 can lead to impaired mitochondrial respiration and energy production along with inhibiting apoptosis through the regulation of BAX activation [8].
Although CHCHD2 involvement in the apoptotic path- way and oncogenesis has been already studied in several others solid tumors such as breast [10, 11], lung [12], renal [13] and pancreatic cancer [14] or even in hematological malignancies [9], no data exist on ACC. Hence, we aimed to study its expression pattern in normal adrenal tissues and to investigate its role in adrenal tumourigenesis.
Methods
Studied population
Twenty-six fresh frozen adrenal tissues were collected prospectively from October 2019 to December 2020 from patients undergoing laparoscopic adrenalectomy either because of tumor size or suspicious imaging characteristics or autonomous hormonal secretion. All surgical procedures were performed by the same surgical team of the 3rd Department of Surgery in the General hospital “G. Genni- matas” in Athens. For every sample of the pathological adrenal tissue in patients proven to be adenomas, the sur- geon collected also a sample of the adjacent adrenal tissue, which was considered macroscopically normal, but not from adrenal tissue next to adrenocortical carcinomas (ACC). The adjacent adrenal tissue was used as “control” only when it was histologically confirmed by the patholo- gist as normal adrenal tissue.
The histopathological analysis of the surgical specimens was performed by two independent pathologists in the Department of Pathology of “G. Gennimatas” and “Evange- lismos” General Hospitals in Athens. All adrenocortical neoplasms were classified according to the universal diag- nostic criteria endorsed by the WHO classifications including the modified Weiss criteria [15]. Malignant adrenal tumors were considered when histopathological analysis demon- strated three or more of the Weiss criteria needed for the diagnosis of ACC. The diagnosis of the autonomous hor- monal secretion from the adrenal neoplasms was based on the history, clinical examination, and preoperative endocrine tests performed in the same laboratory according to ENSAT guidelines. Cortisol (basal and after 1-mg overnight dex- amethasone test (ODST)) and 24-h urinary free cortisol (UFC) levels were measured by electrochemiluminescent bridging immunoassay (ECLIA) (Cobas 8000 e801, Hitachi, intra-assay CV <3.9% and inter-assay CV <3.8% for all hormones), adrenocorticotropic hormone (ACTH) was mea- sured by chemiluminescent assay (Liaison, DiaSorin, intra- assay CV 4.3-7.5% and inter-assay 10-14.5%).
All patients gave written informed consent for the use of samples and clinical data. The protocol of this study was approved by the institutional Research Ethics Board of the National and Kapodistrian University of Athens (Laiko
hospital, code 6545/22-4-2019) according to the guidelines of the Declaration of Helsinki.
Materials
A quantity of 100-200 mg of adrenal tissue (adrenal tumor and normal adrenal tissue) was cut and stored immediately after surgery at -80 ℃ as previously described [16].
Cell culture
The SW13 ACC cell lines (kindly provided by Professor C. Stratakis, NIH, USA) were grown and maintained in high glucose DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% fetal bovine serum and 100 µg/ml penicillin/streptomycin in a standard humidified incubator at 37 ℃ in a 95% air and 5% CO2 atmosphere. Cell monolayers were sub-cultured into a 12-well plate for RNA extraction (4 × 105),for apoptosis analysis using Annexin PI (2 × 105 cells/well) and for wound healing assay (3x 105 cells/well) and into a 96-multi-well plate (103 cells/well) for MTS assay. Cells were maintained in complete medium for 16 h pre- and for 48 h post-transfection.
siRNA
Cells were seeded into 12-multi-well plates (1 x 105 cells/ well) and after 16 h of incubation they were transfected either with control siRNA (siRNA scrambled) or with CHCHD2 siRNA (Thermo Fisher Scientific) using lipo- fectamine 3000 transfection reagent (Thermo Fisher Sci- entific) following the manufacturer’s instructions and optiMEM (complete medium). After 48 h, CHCHD2 levels were detected by RT-PCR and Western Blot analysis.
MTS assay
The effect of CHCHD2 silencing on SW13 cell viability was determined using MTS assay as previously described [17].
Would healing assay
Cells were incubated in 12-well plates until about 70% con- fluent and then they were transfected with CHCHD2 siRNA. After 8 h of transfection, a 10-uL pipette tip was used to create a scratch/wound with clear edges across the well. Cells were photographed with Olympus CKX53 microscope at 0, 24, and 48 h. All experiments were performed in triplicates.
Flow cytometric analysis
Annexin PI: Apoptotic cell death was identified in SW13 cells after CHCHD2 silencing by double supravital staining
with recombinant FITC (fluorescein isothiocyanate) con- jugated Annexin V and propidium iodide (PI), using the Annexin V-FITC Apoptosis Detection kit (4830-01-K) according to the manufacturer’s instructions.
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, Düren, Germany). The purity and concentration of the extracted RNA were determined using the NanoDrop spec- trophotometer 2000 (Thermo Scientific). One microgram of RNA was reverse transcribed into cDNA using a LunaScript® RT SuperMix Kit (New England Biolabs, Ipswich, MA, USA). The mRNA expression of Bax, Bcl-2, CHCHD2 was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) as previously described [14]. Briefly, mRNA levels of the tioned genes were evaluated using Luna® Universal qPCR Master Mix (New England Biolabs, Ipswich, MA, USA) on the CFX96 Touch real-time PCR detection system (Bio-RAD, Hercules, CA, USA). All reactions were carried out in tripli- cates. Each qPCR run included a negative control. The pooled cDNA sample was used as an internal control to correct the inter-assay variation for samples run on different plates. The relative fold change was calculated using the 2(-Delta Delta C(T))(AACTs) after normalizing to the value of internal- control. The expression of each gene for every adrenal sample was normalized against Actin expression [18]. The sequences of primers used in this study are listed below:
| Primer | Sequence |
|---|---|
| Actin | |
| Forward | 5'-AGAGCTACGAGCTGCCTGAC-3' |
| Reverse | 5'-AGCACTGTGTTGGCGTACAG-3' |
| Bax | |
| Forward | 5'- CCGCCGTGGACACAGAC-3' |
| Reverse | 5'- CAGAAAACATGTCAGCTGCCA-3' |
| Bcl-2 | |
| Forward | 5'-GCTGAAGATTGATGGGATCG-3' |
| Reverse | 5'-TACAGCATGATCCTCTGTCAAG-3' |
| CHCHD2 | |
| Forward | 5'-GTGCCGACTTGCAAACGGAT-3' |
| Reverse | 5'-GGCCACACAAACATTTGCCC-3' |
Protein extraction and western blot analysis
Whole 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). Thirty micrograms of extracted protein from each sample were resolved on the 12 or 15% SDS-PAGE gel by electro- phoresis and immediately transferred to PVDF membrane. After blocking with 1X-TBST containing 5% dry milk for 1 h, membranes were incubated overnight at 4 ℃ with anti-b-actin (MAB1501 Millipore; 1:5000), anti-Bax (#5023 Cell Signaling; 1:800), anti-Bcl-2 (#15071 Cell Signaling; 1:800), anti-CHCHD2 (HPA027407 Sigma- Aldrich; 1:1000). Subsequently, membranes were washed 3 times with 1X-TBST and incubated with HRP con- jugated anti-mouse IgG (31430, Thermo Scientific) or anti-rabbit IgG (12-348, Millipore) secondary antibodies for 1 h at room temperature. The protein bands were visualized using the Clarity Western ECL Substrate (Bio- Rad) and quantified using ImageJ software. ß-actin served as a loading control and an aliquot of pooled standard sample was loaded in each gel to correct the inter-assay variation for samples run in different gels.
Statistical analysis
All the data are reported median (IQR). Nonparametric parameters were analyzed with Mann-Whitney test for the comparison between ACCs patients and patients with ACAs or controls while Wilcoxon test was performed for the comparison of nonparametric paired values in the same group. Student t-test was applied to evaluate the difference of parametric parameters between the groups. For corre- lation analysis we used Spearmen rank correlation coeffi- cient test. All tests were 2-sided with statistical significance set at 0.05 and all computation were made using PRISM 7.
Results
Characteristics of the studied population
The study included a total of 26 (18 females) patients pre- senting with a unilateral adrenocortical neoplasm with a median (interquartile range (IQR)) age of 60.25 (12.4) years. All patients underwent unilateral adrenalectomy. Histopathological analysis based on Weiss criteria con- firmed the diagnosis in 16 ACAs and in 10 ACCs. Func- tional tests showed that the 7/16 ACAs consisted of cortisol-secreting adenomas (CSAs) (median size: 6 cm) and 9/16 nonfunctional adrenal incidentalomas (NFAs) (median size: 5 cm) whereas the 10 ACCs consisted of 6 cortisol-secreting and 4 concomitantly cortisol and androgen-secreting tumors. Median survival for ACC patients was 11 months (IQR: 15). The mortality rate was 11.5% (3 out of 26 patients) during a 23 months (IQR: 15.5)
| Adrenocortical neoplasms | CSAs | NFAs | ACCs |
|---|---|---|---|
| Population (n) | 7 | 9 | 10 |
| Age (median, IQR), years | 60.5(6) | 61(19) | 48(27) |
| Sex(F/M) | 6/1 | 6/3 | 6/4 |
| -Hormonal profile | |||
| Mean±SD baseline F levels (08:00am) | 23 ±8.8 | 19±2 | 25 ±13 |
| Mean ± SD UFC/24 h. levels | 165±106 | 79.4 ±16.7 | 250 ±78 |
| Mean ± SD F post-1mg | 4.2±2.1 | 1.2±0.43 | 10.9±3.4 |
| ODST levels | |||
| -Tumor characteristics | |||
| Size(median, IQR), cm | 6(1.12) | 5(2.75) | 6(5.8) |
| Ki-67% | – | – | 15(14%) |
| Weiss(median, IQR) | – | – | 6(3) |
| Survival (IQR), months | – | – | 11(15) |
| Mortality rate(%) | – | – | 3/26(11.5%) |
| Follow-up (IQR), months | – | – | 23(15.5) |
CSAs cortisol-secreting adenomas, NFAs Nonfunctional adenomas, ACCs adrenocortical carcinomas, F/M Female/Male, IQR interquartile range
median follow-up, whereas 4/10 patients were metastatic at diagnosis. Clinical, biochemical, and histological data are presented in Table 1.
Genes’ mRNA and protein expression in adrenocortical neoplasms
mRNA and protein expression of BAX, BCL2 in adrenocortical neoplasms
BCL2 mRNA expression (fold change) was found to be upregulated in all adrenocortical neoplasms compared to the adjacent normal adrenal tissue (paired control samples from ACAs). In contrast, BCL2 mRNA levels were sta- tistical significantly upregulated in all ACCs samples compared to ACAs (p =0.047) and adjuvant normal adrenal tissues (p=0.025) (Fig. 1A). BCL2 mRNA expression was also upregulated in all ACAs (CSAs and NFAs) compared to their paired normal adrenal tissue, albeit without obtaining a statistical significance (p = 0.405) (Fig. 1A). However, mRNA differences were not confirmed at protein level since no l significant dif- ferences in protein levels were found among ACCs, ACAs (in total as well as separately in CSAs and NFAs) and normal adrenal tissue (Figs. 1B, C, and 2A).
On the contrary, BAX mRNA and protein expression was downregulated in all adrenocortical neoplasms (ACCs and ACAs) compared to normal adrenal tissue (paired control samples from ACAs) (Fig. 1A-C). In particular, BAX
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mRNA and protein expression were statistically sig- nificantly downregulated in all ACCs compared to the normal adrenal tissue (p=0.027 and p=0.002, respec- tively) as well in ACAs (p=0.049 and p=0.0019 for mRNA and protein levels, respectively) (Fig. 1A, B). BAX mRNA and protein levels were also downregulated in all ACAs (CSAs and NFAs) compared with their paired nor- mal adrenal tissues although not statistically significantly (p=0.689 and p=0.08, respectively) (Fig. 1A). When analyzed separately, BAX protein expression appeared to be lower in CSAs and NFAs compared with their paired nor- mal adrenal tissue (p=0.1 for CSAs and p=0.03 for NFAs) (Fig. 2B). Moreover, BAX protein levels were sig- nificantly lower in ACCs compared with CSAs (p =0.009) as well as NFAs (p=0.003) (Fig. 3B).
Neither BCL2 nor BAX mRNA and protein expression showed a significant difference between CSAs and NFAs (p<0.5) (Figs. 1B and 3).
mRNA and protein expression of CHCHD2 in adrenocortical neoplasms
CHCHD2 mRNA and protein expression were upregulated in all adrenocortical neoplasms (ACCs and ACAs) com- pared with normal adrenal tissues (paired control samples from ACAs) (Fig. 1A, C). CHCHD2 mRNA and protein expression was significantly upregulated in all ACCs sam- ples compared with the normal adrenal tissues (p = 0.05 and p = 0.024, respectively) (Fig. 1A, C). Moreover, CHCHD2 protein expression was also significantly upregulated in all ACCs compared with the ACAs (p=0.029) samples although this didn’t reach statistical significance in mRNA levels (p=0.212) (Fig. 1A, C).
Of note, when analyzing separately ACAs, CHCHD2 mRNA and protein expression were higher in ACCs com- pared with NFAs and CSAs samples although statistically significant only at the protein levels (p=0.04 and
2.A.
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p = 0.009, respectively). CHCHD2 mRNA and protein expression showed no difference between CSAs and NFAs (p=0.865) (Figs. 1B and 3).
The prognostic role of genes’ expression in adrenocortical neoplasms
CHCHD2 mRNA expression was negatively correlated with overall survival (OS) and progression-free survival (PFS) (r =- 0.86, p=0.6 and r =- 0.5, p=0.9, respectively) in ACC patients. Similarly, BCL2/ BAX ratio mRNA expression was negatively correlated with OS and PFS (r =- 0.5, p=0.9 and r =- 0.3, p=0.7, respectively) without though reaching statistical sig- nificance. The same pattern is also found in protein levels without obtaining any statistical significance. Patients who succumbed to the disease had a five-fold higher ratio (median mRNA ratio fold: 5.5) of CHCHD2 mRNA expression compared to survivors (median mRNA ratio fold: 0.26). No association was found between any
of the three studied genes CHCHD2, BAX, and BCL2 with Ki-67 and Weiss score at both mRNA and protein levels.
In vitro analysis in SW13 cells
CHCHD2-siRNA transfection suppressed the proliferation and induced cell apoptosis of SW13
The efficiency of silencing was evaluated by qPCR and western blot analysis (Fig. 4A, D). The effect of CHCHD2 in silencing SW13 cells’ proliferation was evaluated by MTS assay. It was demonstrated that the proliferation of CHCHD2 in silencing SW13 cells was significantly decreased both time- and dosage-dependently. Cell viability was significantly reduced at a concentration of 20 um after 24 h of transfection, as well as at concentrations of 10 and 20 um 48h post-transfection (p<0.05 and p<0.01, respectively) compared with the cells transfected with nontargeted siRNA (Fig. 5). The cell proliferation’s
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inhibition rate at the highest concentration was 20.56 and 32.46% after 24 and 48 h of incubation, respectively.
CHCHD2-siRNA induced apoptotic rate of SW13 cell through BAX overexpression
The apoptotic rates of SW13 cells after transfection with either CHCHD2-siRNA or NT-siRNA were determined by the Annexin V-propidium iodide through flow cytometry method. Our results demonstrated that SW13 cell’s apop- tosis was 2.5 times increased after CHCHD2 silencing as compared to cells transfected with nontargeted siRNA (Fig. 4C). qPCR analysis showed that cell’s apoptosis was induced at least in part, after CHCHD2 silencing associated with significant increase of BAX (p<0.05) mRNA levels and consequent reduction of BCL2/BAX ratio (p<0.05). These results were further confirmed at the protein levels (Fig. 4D).
CHCHD2-siRNA inhibits SW13 cell’s invasion in vitro
Scratch assays showed that SW13 cell invasion was sig- nificantly reduced after CHCHD2 siRNA transfection as compared to cells transfected with nontargeted siRNA (NT- siRNA) (p<0.01). These results suggest that CHCHD2 silencing may reduce the SW13 cells migration and inva- sion (Fig. 5).
Discussion
This study provides evidence that the expression of the apoptosis-regulated genes BAX, BCL2, and CHCHCD2 is altered in adrenocortical neoplasms compared to the adja- cent normal adrenal tissue. BAX was found to be down- regulated whereas BCL2 and CHCHD2 were upregulated in all adrenocortical neoplasms. In particular, BCL2 and CHCHD2 were both significantly upregulated in all ACCs compared to normal adrenal tissue and ACAs. In vitro analyses in transfected SW13 cells showed that CHCHD2 silencing resulted in reduced cell viability and invasion as well as increased SW13 cell apoptosis in line with the data from human samples. Although, no association of the expression of the studied genes with other prognostic markers of disease progression was found in patients with ACCs, CHCHD2 mRNA expression was five-fold upregu- lated in nonsurvivors ACC patients compared with survi- vors. Although this finding was highly significant the small number of patients studied should be regarded with caution until further studies with larger number of patients are performed.
Data regarding the role of the apoptotic pathways in adrenal tumorigenesis are extremely rare. Higher expression of BCL2 and BCL-XL in ACCs and ACAs compared to normal adrenal surgical specimens have been reported in several studies that utilized mostly through immunohistochemical or
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RT-PCR analysis [19-23]. Moreover, BAX gene expression was absent in ACCs specimens, whereas it was present in ACAs and normal adrenal glands implying its potential role in apoptosis in ACCs [23]. Recently, an in vitro study demon- strated that CHCHD2 was able to inhibit apoptosis in human cancer cells by interacting with BCL-XL thus regulating BAX
activation [8]. Further data have demonstrated that activation of BCL2 cleaved BAX, releasing caspase -9 and -3 that are involved in the intrinsic apoptosis pathway, eventually indu- cing SW13 cell apoptosis [24]. In line with previous studies, our results showed that BCL2 expression, albeit not at the protein level, was found significantly upregulated and BAX
downregulated in all ACCs samples compared to ACAs and normal tissues. Moreover, CHCHD2 upregulation in all ACCs tissues samples has demonstrated a potential regulatory role in ACCs cell apoptosis through the BCL2/BAX pathway [8]. Indeed, in vitro analysis silencing of CHCHD2 in SW13 cells confirms this hypothesis as it was associated with increased apoptosis, reduced cell viability, and increased BAX mRNA expression implying that CHCHD2 overexpression may pro- mote ACC spread and metastasis to distance organs.
More specifically, there is evidence of the prognostic role of CHCHD2 in other malignancies their metastatic status and survival. CHCHD2 immunohistochemical expression was found significantly higher in the nonsmall cell lung cancer tissues of patients with lymph nodes metastasis (p<0.05) compared to the nonmetastatic ones [25]. Simi- larly, CHCHD2 was found to be correlated (overexpressed) with the presence of distant metastasis in breast cancer patients (p<0.05) and its overexpression was associated with worse OS [11]. CHCHD2 immunohistochemical expression was also shown to be higher in hepatocellular carcinoma tissues of patients with lymph node metastasis, and higher TNM grade and was associated with worse patients’ survival [26]. In the same study, in vitro data demonstrated that depletion of CHCHD2 led to significantly reduced migratory capacity of HepG2 cells. In agreement to the last finding, further in vitro data showed that CHCHD2 knockdown inhibited renal cell carcinoma migration and tube formation of human umbilical vascular endothelial cells [13].
On the contrary, data regarding the role of apoptosis- related genes on ACC prognosis are extremely limited. One study had demonstrated that Bok gene encoding BCL2 family protein was overexpressed in ACC specimens of patients with poor prognosis [19]. Furthermore, BCL2 protein expression in surgical adrenal tumoural specimens was negatively correlated with tumor size, advanced disease stage, and survival analyzed, however, only through immunohistochemistry [27, 28]. Our series didn’t show any significant association of CHCHCD2 expression in ACC samples with established prognostic markers, Ki-67% index, and Weiss score, considering though that the sample size was too small for robust conclusions to be made.
CHCHD2 protein has been shown to play a role in the regulation of cell metabolism and the inhibition of apop- tosis. Recent research has shown that CHCHD2 can disrupt oxidative phosphorylation and mitochondrial metabolism and promote cell migration through an AKT-dependent mechanism. In addition, CHCHD2 has been found to be involved in cytoskeleton and membrane trafficking [29, 30]. It has been also suggested that CHCHD2 may function as an antioxidant to remove reactive oxygen species as well as an activator of BCL-XL, which are both involved in pro- moting cell survival [29, 30]. The interaction of CHCHD2
with BCL2/BAX in the apoptotic mechanism has not been fully clarified. The mechanism of apoptosis is based on MOMP which is controlled by BCL2 family proteins, such as BAX which is activated and oligomerizing in response to various apoptotic stimuli [31]. BAX oligomerization helps release mitochondrial contents into the cytoplasm, which activates the caspase cascade. BCL-XL, is another anti- apoptotic BCL2 protein primarily regulating BAX and preventing its accumulation at the mitochondria. Activation of BCL2 cleaved BAX, releasing caspase-9 and -3 involved in the intrinsic apoptosis pathway, eventually inducing SW13 cell apoptosis [24].CHCHD2, a small mitochondrial protein binds to BCL-XL and inhibits the mitochondrial accumulation and oligomerization of BAX. A recent in vitro study showed that when cells are exposed to apoptotic sti- muli, the mitochondrial levels of CHCHD2 decrease before MOMP occurs [8]. Furthermore, CHCHD2 absence from the mitochondria, reduces BCL-XL’s ability to inhibit BAX activation resulting in increased BAX oligomerization, MOMP, and ultimately apoptosis. Indeed our data showed that in vitro analysis silencing of CHCHD2 in SW13 cells resulted in increased BAX mRNA expression [8]. Overall, these findings suggest that CHCHD2 may play a beneficial survival role in cells by regulating various cellular pro- cesses. Mitochondrial-mediated therapeutics in cancer can be achieved through targeting various involved pathways inside the mitochondria. In a study, a monocarboxylate transporter MCT1/2 inhibitor (AR-C155858) reduced the mammospheres formation of breast cancer cell lines (MCF7 and T47D cell lines) [29].
Several in vitro and in vivo studies have presented data of alternative therapeutic agents that may potentially improve prognosis in ACC patients either by targeting proteins that prevent apoptosis or by activating molecules that promote it [32-34]. It has been already shown that gossypol -a compound that inhibits BCL2 and BCL-XL - resulted in partial response in 3 patients with advanced ACC resistant to chemotherapy and stable disease in another one. [35]. More recent studies found that gossypol has two stereoisomers, horwever, the (-) stereoisomer has the highest affinity for BCL2 and BCL-XL [36]. The com- bination of (-) gossypol and docetaxel resulted in a greater inhibition of the cell proliferation in a dose-dependent manner in the human adrenal cancer cell line RL-251 that expresses high levels of BCL-XL, compared to the cell line H-295 with low levels of BCL-XL as well as a complete growth suppression of ACCs with high expression of BCL- XL. Thus, it was suggested that BCL-XL expression may determine the response to pro-apoptosis drugs [37].
The small number of our sample, the use of only one cell line as well as the lack of data regarding the CHCHD2 expression in SW13 are the main limitation of this study, nevertheless, the scarce data on the role of apoptotic
mechanisms in the adrenal tumorigenesis rends this study novel with potentional interesting clinical implications.
Conclusions
In conclusion, we showed that similarly to other tumors, CHCHD2 mRNA and protein levels were overexpressed in adrenocortical neoplasms compared to adjacent normal adrenal tissue showing a significant overexpression in ACCs along with alteration of BCL2/BAX expression. Furthermore, CHCHD2 knockdown in SW13 cells showed increased apoptosis, reduced cell viability and increased BAX expression confirming the involvement of CHCHD2 in the regulation of cell apoptosis in ACC Further studies in larger number of patients are needed to confirm these data and delineate the role of CHCHD2 into the immune- metabolic and cytogenic pathways might lead to the design of combination targeted therapeutic regimens.
Author contributions All authors contributed to the study conception and design: Initial conceptualization by AA. Material preparation, data collection and analysis including experiments were performed by AK, NN-A, AM, GCT, GK, TC, AP, GZ, CA. The first draft of the manuscript was written by AA. GK and EK edited and commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Funding This work was supported by the National and Kapodistrian University of Athens and the Empeirikion Institution (grant number 15711, 10/05/2018).
Compliance with ethical standards
Conflict of interest The authors declare no competing interests.
Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by B the insti- tutional Research Ethics Board of the National and Kapodistrian University of Athens (NKUA) (Laiko hospital, code 6545/22-4-2019).
Consent to participate Informed consent was obtained from all indi- vidual participants included in the study.
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