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Vitamin D receptor activation is a feasible therapeutic target to impair adrenocortical tumorigenesis
Ana Carolina Buenoª, Candy Bellido More ª, Junier Marrero-Gutiérrez b, Danillo C. de Almeida e Silvab, Leticia Ferro Lealª,1, Ana Paula Montaldi ”, Fernando Silva Ramalho ª, Ricardo Zorzetto Nicoliello Vêncio e, Margaret de Castrob, Sonir Roberto R. Antonini
a Department of Pediatrics, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP, Brazil
b Department of Internal Medicine, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP, Brazil
” Department of Biology, Faculty of Philosophy, Sciences and Letters at Ribeirao Preto, University of São Paulo, Ribeirao Preto, SP, Brazil
d Department of Pathology, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, SP, Brazil
e Department of Computation and Mathematics, Faculty of Philosophy, Sciences and Letters at Ribeirao Preto, University of São Paulo, Ribeirao Preto, SP, Brazil
ARTICLE INFO
Keywords:
Adrenocortical tumor Vitamin D receptor
H295R cells
Target therapy
ABSTRACT
Objective: To evaluate the therapeutic potential of vitamin D receptor (VDR) signaling in adrenocortical carci- noma (ACC) cells.
Methods: We evaluated VDR’s methylation pattern in H295R ACC cells, and investigated the effects of calcitriol and seocalcitol treatments on adrenocortical tumorigenesis.
Results: VDR was hypermethylated and underexpressed in basal H295R cells. Treatments with calcitriol and seocalcitol restored VDR signaling, resulted in antiproliferative effects, and impaired Wnt/B-catenin signaling. RNAseq of treated cells demonstrated VDR activation on steroid hormones biosynthesis and Rap1 signaling, among others. In vivo, seocalcitol constrained the growth of H295R xenografts and reduced autonomous tumor steroid secretion without hypercalcemia-associated side effects.
Conclusions: H295R cells present VDR hypermethylation, which can be responsible for its underexpression and signaling inactivation under basal conditions. VDR signaling promoted antiproliferative effects in vitro and in vivo, suggesting that it may be a potential therapeutic target for ACC and a valuable tool for patient’s clinical management.
1. Introduction
Adrenocortical carcinomas (ACC) are a rare malignancy that occurs most frequently in the first 5 years of age and around the 50th decade of life. Pediatric and adult ACC present distinct epidemiological, clinical, histopathological, molecular features and survival rates. In children, there is an exceptionally increased incidence of adrenocortical tumors (ACT) in Southern Brazil. These tumors are frequently diagnosed due to clinical manifestations of excessive hormone secretion, and present better prognoses. In adults, ACC often present as steroid hormone- producing tumors (>80% of cases), with variable prognosis (Fassnacht
et al., 2013; Mansmann et al., 2004; Rodriguez-Galindo et al., 2005).
Irrespectively of the age at onset, complete surgical tumor resection is the only curative treatment for patients with localized disease (Fass- nacht et al., 2013; Tucci et al. 2005). For those with advanced or met- astatic disease, the use of systemic adjuvant therapies results in limited improvement of survival (Fassnacht et al., 2012; Megerle et al., 2018). Hence, there is an effort for the development of novel target-specific therapies for these patients (Mohan et al., 2018).
Aberrant Wnt/B-catenin is a hallmark of adrenocortical tumorigen- esis and a predictor of unfavorable outcomes for pediatric and adult patients (Gaujoux et al., 2011; Leal et al., 2011; Mermejo et al., 2014;
* Corresponding author. Ribeirao Preto Medical School - University of Sao Paulo, Avenida Bandeirantes, 3900 Monte Alegre, CEP14049-900, Ribeirao Preto, Sao Paulo, Brazil.
E-mail address: antonini@fmrp.usp.br (S.R.R. Antonini).
Current affiliation: Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, SP Brazil; Barretos School of Health Sciences, Dr. Paulo Prata - FACISB, Sao Paulo, Brazil.
https://doi.org/10.1016/j.mce.2022.111757
Tissier et al., 2005). Whereas preclinical studies showed promising re- sults on the inhibition of the Wnt/B-catenin signaling in ACC cells (Doghman et al., 2008; Gaujoux et al., 2013; Leal et al., 2015; Salomon et al., 2015), early phase clinical trials struggle with therapeutic tissue selectivity and important side effects (Shaw et al., 2019).
The vitamin D receptor (VDR) is ubiquitously expressed in the endocrine system. Upon heterodimerization with the retinoid X receptor (RXR), VDR signals in a cell-specific manner, reliant on an of complex genomic machinery in order to mediate the expression of numerous target-genes (Baker et al., 1988; Lee et al., 2015; Pike et al., 2016; Zella et al., 2010). Several clues link normal VDR signaling with adrenal ho- meostasis and steroidogenesis. Recently, it was demonstrated that VDR nuclear expression is present in normal (non-pathological) human ad- renal cortex throughout adrenal development (Bueno et al., 2022), and in the cells from zona fasciculata, zona reticularis and, mainly, in zona glomerulosa of mature adrenals (Gao et al., 2019). Preclinical studies demonstrated downregulation of 21-hydroxylase (CYP21A2) and repression of glucocorticoid and mineralocorticoid synthesis in H295R adrenocortical cells treated with calcitriol (1,25-Dihydroxivitamin D3), which is the active metabolite of vitamin D3 and canonical ligand to VDR (Lundqvist et al., 2010, 2012).
The antiproliferative effects of VDR signaling were demonstrated in different types of cancers, and some calcitriol analogs yield higher anti- tumor activity and lesser effects on calcium and phosphate homeostasis when compared to calcitriol (Gulliford et al., 1998; VanWeelden et al., 1998; DeLuca and Plum, 2016). Of note, it was demonstrated that VDR transactivation represses B-catenin signaling and tumor proliferation in different cancers that progress with aberrant Wnt/B-catenin signaling (Aguilera et al., 2007; Beildeck et al., 2009; Johnson et al., 2015; Mur- alidhar et al., 2019).
Increased VDR gene promoter methylation and reduced expression were demonstrated in ACC from adult patients (Pilon et al., 2014, 2015). We have recently uncovered VDR’s underexpression in pediatric ACT, especially in ACC. Moreover, using whole-genome methylation analysis, we showed that VDR hypermethylation associates with reduced mRNA levels and with unfavorable outcomes for children (Bueno et al., 2022). Altogether, these observations indicate that ACC have impaired VDR expression - at least in part due to hypermethylation - irrespective of the age of disease onset.
In the present study, in order to assess whether vitamin D3 plays antiproliferative effects in adrenocortical tumorigenesis, we used the human ACC H295R cell line, which harbor constitutive abnormal Wnt/ B-catenin activation (Tissier et al., 2005), and demonstrate the thera- peutic potential of VDR signaling activation.
2. Materials and methods
2.1. In vitro study
This study was performed in the Laboratory of Molecular Endocri- nology of the Ribeirao Preto Medical School - University of Sao Paulo (FMRP-USP) University Hospital and was approved by the Institution’s Ethics Committee (#343/758).
2.1.1. Cell culture
Human H295R (RRID:CVCL_0458) ACC cell lines were subcultured, authenticated by STRS DNA profiling, and genotyped for the exon 3 CTNNB1 (B-Catenin coding gene) p.S45P mutation, as previously described (Leal et al., 2015).
2.1.2. VDR methylation profiling of H295R cells
DNA was extracted from subcultured H295R cells using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufac- turer’s instruction. DNA was quantified by fluorometry using the Qubit dsDNA BR Assay (Thermo Fisher Scientific, Waltham, MA), and the integrity was assessed by electrophoresis using the Tape Station 4200
System (Agilent, Santa Clara, CA). VDR DNA methylation profiling was assayed using the Human Infinium Methylation EPIC BeadChip Array (Illumina, San Diego, CA) as previously described (Bueno et al., 2022). Briefly, the methylation data from 53 probes targeting the VDR gene and its immediate vicinity were retrieved. Methylation levels were evaluated using the M-value: a log2 ratio between methylated and unmethylated versions of the probes. VDR methylation data from H295R cells (Sup- plementary Table 1) were combined with those from our adrenocortical tumor cohort (Bueno et al., 2022) and subjected to an unsupervised hierarchical clustering analysis (UHCA), using R statistical language’s (RRID:SCR_001905) Pvclust Bioconductor package (RRID:SCR_021063) to perform Ward’s clustering with Euclidean distance and 2000 boot- strap resampling runs to access robustness.
2.1.3. Treatment with vitamin D3
The vitamin D3 active metabolite calcitriol and its synthetic analog seocalcitol (Cat#D1530 and Cat#SML1727, respectively, Sigma- Aldrich, St Louis, MO, USA) were resuspended in ethanol (vehicle) at stock concentrations of 10-4 M and diluted in complete growth medium according to the required concentrations (10-9 to 10-6 M). Twenty-four hours after seeding, the H295R cells were treated with calcitriol, seo- calcitol or the correspondent amount of vehicle (control). After the depicted time points, the cells were processed for different assays, as following described.
2.1.4. Cell proliferation
Cell viability was assessed after seeding 2 × 104 cells per well in 96- well plates and treating them with increasing doses of calcitriol, seo- calcitol or vehicle for up to 96 h. The CellTiter AQueous One Solution Proliferation Assay (Cat#G3581, Promega Corporation, Madison, WI, USA) was used according to the manufacturer’s instructions. Three in- dependent experiments were carried out in triplicate.
Cell cycle analysis was performed after seeding 2 × 105 cells per well in 24-well plates and treating them with calcitriol (10-7 M) or vehicle for 48 and 96 h. The cells were fixed in ice-cold ethanol overnight at -20 ℃, and then stained with a solution containing 5 µg/mL propidium iodide and 50 µg/mL ribonuclease A (RNase) (both from Sigma Aldrich). The cell’s DNA content was measured using flow cytometry on a Guava Personal Cell Analysis system (Guava Technologies, Hayward, CA, USA), according to the manufacturer’s protocol. The percentages of cells in the Sub-G1/Apo, G0/G1, S, or G2/M phases were collected and processed using the Guava CytoSoft 4.2.1 version software. Three independent experiments were carried out in triplicate.
2.1.5. RNA extraction and quantitative real-time PCR
Total RNA from cell lines and animal tissues (subsection 2.2) was extracted using the TRIzol® Reagent and quantified by spectrometry (Nanodrop 2000; Thermo Fisher Scientific Inc. Waltham, MA, USA) at 260 nm. RNA integrity was checked according to the 260/280 nm ratio with an acceptable range of 1.6-2.0 and confirmed by 1.2% agarose gel electrophoresis. RNA samples (500 ng) were reverse transcribed using the High-Capacity cDNA Reverse Transcription kit and MultiScribe® enzyme (Life Technologies). Real-time qPCR was performed using TaqMan® assays (Applied Biosystems™ ThermoFisher Scientific). For H295R cells: AXIN1 (Hs00394718_m1), AXIN2 (Hs00610344_m1), CYP24A1 (Hs00167999_m1), CYP27B1 (Hs01096154_m1), CTNNB1 (Hs00170025_m1), CCND1 (Hs00765553_m1), CCNE1 (Hs01026536_m1), CDK4 (Hs00175935_m1), CDK2 (Hs01548894_m1), DKK3 (Hs00951307_m1), MYC (Hs00153408_m1), SFRP1 (Hs00610060_m1), TCF7 (Hs0017273_m1), VDR (Hs00172113_m1), and GUSB (4326320 E) as endogenous control, as previously described (Abduch et al., 2016). For murine kidney samples: Cyp27b1 (Mm01165918_g1), Cyp24a1 (Mm00487244_m1), Vdr (Mm00437297_m1), and Actb (4352933) and Gapdh (4352932) were used as endogenous controls. Relative mRNA expression levels were calculated using the using the 2-AACt method.
2.1.6. RNA-seq
Total RNA samples from H295R cells treated with calcitriol or seo- calcitol (10-7 M, three biological replicates per treatment), or vehicle (two biological replicates) for 48 h were extracted as described in sub- section 2.1.5. Quantification was performed by fluorometry using the Qubit RNA BR Assay (Thermo Fisher Scientific, Waltham, MA). Integrity was assessed by electrophoresis using the Tape Station 4200 System (Agilent, Santa Clara, CA) and RNA integrity number (RIN) ≥9 was considered adequate.
One microgram of RNA was used to prepare the RNA library, to convert cDNA, and to select poly-A, according to the Illumina’s Stranded mRNA Prep instructions. Sequencing was performed on Illumina’s HiSeq 4000 RNA-seq platform (Illumina) with paired-end libraries and frag- ments of 300 bp (2 x 150 bp).
RNA-Seq analysis was performed according to the following pipeline. All sequencing reads were converted to FASTQ files. The sequencing adapters were removed, and the quality was checked using the FASTQC program (v. 0.11.9, RRID:SCR_014583) (Andrews, 2010). We removed the duplicated sequences using the SeqKit tool (v. 2.2.0) (Shen et al., 2016), the first 10 bases of the reads and kept the bases with a sequencing quality greater than 30 at the beginning and end of the reads, using the trimmomatic tool (v. 0.39, RRID:SCR_011848) (Bolger et al., 2014). Reads were pseudo-aligned to the human genome version GRCh38 using kallisto (v. 0.46.0, RRID:SCR_016582) (Bray et al., 2016), considering the bias of genomic GC content and 100 bootstrap-sampling in the reliability of the abundance estimates. Transcription-level abun- dances, estimated counts and transcription lengths of each sample were subsequently imported into the R environment with the tximport tool (RRID:SCR_016752) (Soneson et al., 2015), which were used to deter- mine differentially expressed genes (DEGs) using the DESeq2 package (RRID:SCR_015687) (Love et al., 2014) . We consider DEGs if the false discovery rate, controlled by Benjamini-Hochberg’s procedure (pFDR), was <0.01 and the absolute value of log2 fold change >0.75 (|log2FC| ≥0.75). Such threshold was established in order to enclose VDR differ- ential expression between VD3-treated and control H295R cells. To support the visualization of DEGs we created volcano-plots using the ggplot2 package (RRID:SCR_014601) (Wickham, 2016) and heatmaps using Euclidean distance and Ward’s criterion as a clustering method to observe the changes in gene read-counts by sample, as a function of average expression determined by normalized read counts (rlog — transformed) (Gu et al., 2016). Gene ontology (GO) terms (biological functions and processes) and pathway enrichment were investigated by over-representation analysis (ORA) of the DEGs (Yu et al., 2012) considering the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (Homo sapiens). Bar- and cnet-plots of enriched terms and pathways were also created using the aforementioned ggplot2 package in R.
2.1.7. Protein isolation and western blot
H295R cells (1 × 106 per well) were seeded in 6-well plates and treated with calcitriol (10-7 M) or vehicle (72 h). Nuclear and cyto- plasmic protein fractions were extracted using the CelLytic™M Nu- CLEAR™M Extraction Kit (Cat#NXTRACT, Sigma-Aldrich) using Protease Inhibitor Cocktail (P8340, Sigma-Aldrich). Equal amounts of protein (30 µg) were separated (SDS-PAGE) and subjected to immunoblotting. Pri- mary antibodies included anti-VDR (1:500, Santa Cruz Biotechnology Cat# sc-1008, RRID:AB_632070), anti-B-catenin (1:2000, BD Bio- sciences Cat# 610154, RRID:AB_397555), anti-B-actin (1:1000, Santa Cruz Biotechnology Cat# sc-81178, RRID:AB_2223230) and anti- fibrillarin (1:1000, Cell Signaling Technology Cat# 2639, RRID: AB_2278087). HRP-conjugated secondary antibodies included: anti- mouse IgGk BP (Cat# sc-516102, RRID:AB_2687626) and anti-rabbit (Cat# sc-2357, RRID:AB_628497) (both 1:4000; Santa Cruz Biotech- nology). The complexes were developed by enhanced chem- iluminescence (ECL Prime Western Blotting Detection Reagent, GE Healthcare) on Bio-Rad Chemidoc XRS Gel Imaging System (RRID:
SCR_019690). Two independent experiments were performed in tripli- cate for each treatment.
2.1.8. Immunofluorescence
H295R cells (2 x 105 per well) were seeded on coverslips in 24-well plates and treated with calcitriol or seocalcitol (10-7 M), or vehicle (48 h). The cells were fixed in 4% paraformaldehyde, permeabilized in 0.25% Triton X-100 PBS, and blocked with 10% normal horse serum (Gibco, Cat#26050070). Cells were incubated with the aforementioned primary antibodies (subsection 2.1.5) against VDR (1:250) and B-cat- enin (1:2000) overnight at 4 ℃. Immunofluorescent staining was detected using the secondary antibodies goat anti-rabbit IgG FITC (1:1000; Abcam Cat# ab6018, RRID:AB_955224) and CyTM5- conju- gated AffiniPure donkey anti-mouse (1:250; Jackson ImmunoResearch Labs Cat# 715-175-150, RRID:AB_2340819). Nuclear staining was performed using DAPi (1:25000; Cell Signaling Technology Cat# #4083) and the slides were set with ProLongTM Diamond Antifade Mountant (Thermo Fisher Scientific Cat# P36961). Fluorescence was acquired with an Imager.A1 fluorescence microscope (Zeiss) using the AxioVision Imaging System (RRID:SCR_002677) with fixed exposure time for all samples. Three independent experiments were performed in duplicate for each treatment.
2.2. In vivo study
This segment of the study was conducted in the Laboratory of Experimental Animal Studies at the Blood Center Foundation in Ribeirao Preto following the ARRIVE (RRID:SCR_018719) guidelines. The study was approved by the FMRP-USP Ethics Committee on Animal Experi- mentation (#196/2017) and by the National Biosafety Technical Com- mittee (CTNBio, #297/2017.016.01).
2.2.1. H295R xenografts
The effects of seocalcitol on ACC proliferation were investigated using 8-week-old female athymic NOD/SCID-gamma (NSG) mice (IMSR Cat# JAX_005557, RRID:IMSR_JAX:005557). The mice were housed under controlled temperature (22-25 ℃) and 12 h light-dark cycle. Water and food were provided ad libitum. Standard irradiated chow (Nuvilab CR-1; Nuvital® Nutrientes LTDA, Curitiba, PR) containing 1.2% calcium and 2.000 UI/kg vitamin D3 was used throughout the experiment. H295R cells were subcultured, pelleted and washed with DPBS (Sigma-Aldrich) for three times. The cells were then resuspended in DPBS:Matrigel [(1:1), BD Biosciences Cat#354262] solution at a concentration of 2.5 x 10° cells per 100 uL and injected subcutaneously (100 µL) into both flanks of the mice. Body weight and tumor growth were monitored weekly. Tumor volumes (V) were assessed using a caliper (Starrett) by measuring the largest (D, mm) and the smallest (d, mm) perpendicular diameters, and were calculated using the formula: V (mm3) = (D x d2)/2.
2.2.2. Seocalcitol treatment
The treatment with 0.8 µg/kg seocalcitol (equivalent to 4.5 nmol/ mouse/day, n = 5 mice) or vehicle (80% propylene glycol-20% PBS, n = 5 mice) began once the H295R xenografts were established (V = 100 mm3). The mice were daily treated with subcutaneous injections (100 „L), administered 5 times per week during 5 weeks. One week after the end of the treatments the mice were anaesthetized and sacrificed. Car- diac puncture was performed, blood samples were collected, and the serum was isolated by centrifugation, aliquoted, and stored at -20 ℃ until further analysis. The xenografts were excised, weighted and stored accordingly for further analysis. In addition, one kidney from three animals per group was collected and immediately frozen in liquid ni- trogen for mRNA extraction and quantification.
2.2.3. Histological analysis
The excised xenografts were immediately fixed in 4% formalin. The
samples were transversally sectioned into 5-um thick sections, mounted on glass slides, and stained with hematoxylin-eosin (H&E). An experi- enced pathologist analyzed the tissue sections’ histology.
2.2.4. Serum calcium and steroid evaluation
Calcium serum levels were determined by colorimetry (Cayman Chemical, Ann Arbor, MI Cat#701220). Autonomous xenograft steroid secretion was evaluated by measuring mice testosterone and cortisol serum levels by RIA as previously described (Moreira and Elias 1992).
2.3. Statistical analysis
Continuous variables were reported individually, and/or collapsed (mean ± SEM or median), as informed in figure legends. Appropriate statistical tests were used accordingly: Mann-Whitney, Kruskal-Wallis, Student’s t-test, ANOVA, or Pearson’s correlation test. Dunnett’s mul- tiple hypothesis test correction method was used when appropriate. GraphPad Prism software (v.9.0.0, RRID:SCR_002798) was used for statistical analyses. The minimum statistical significance level was set at p ≤ 0.05.
3. Results
3.1. In vitro study
3.1.1. VDR methylation in H295R ACC cells
Unsupervised hierarchical clustering of VDR methylation data from H295R cells with our pACT cohort revealed a High VDR methylation signature. H295R cells clustered within the High VDR methylation group, more exactly in the branch (#51) that holds the tumors with the highest methylation levels, and in the arm (#48) composed only by ACC (Fig. 1a, Supplementary Table 2).
3.1.2. VDR expression and activation in H295R ACC cells
We retrieved the VDR mRNA levels from our pACT cohort and compared them with the mean level from H295R cells grown under basal conditions according to methylation grouping. H295R cells had the far inferior VDR mRNA level in its methylation ACC arm, as well as when compared to the other pACT samples (Fig. 2a). However, upon treatment with calcitriol (10-7 M; 48 h), VDR mRNA levels (p <0.0001; Fig. 2b) and nuclear expression (Fig. 2c and d) were significantly increased in H295R cells. Calcitriol also affected the expression of two canonical VDR targets: it down-regulated CYP27B1 (p = 0.04) and propelled CYP24A1 expression, which was absent under basal condi- tions (Fig. 2b and e). Seocalcitol (10-7 M; 48 h) also successfully increased VDR (p= 0.005), decreased CYP27B1 (p= 0.01) mRNA levels (Fig. 2f), stimulated CYP24A1 transcription (Fig. 2g), and promoted VDR nuclear accumulation (Fig. 2c). These results demonstrated that VDR signaling is deactivated in H295R cells, but the treatment with vitamin D3 active metabolite and its analog are able to restore it. In addition, another observed effect of calcitriol treatment in these cells was the change in their morphology from spindle to rounded-shaped (Supplementary Fig. 2).
3.2. Effects of VDR activation in H295R ACC cell proliferation
To explore the effects of VDR activation on ACC proliferation, we treated H295R cells with increasing doses of calcitriol and analyzed cell viability and cell-cycle progression. Cell viability was significantly decreased with a starting dose of 10-7 M of calcitriol after 96 h of treatment (p = 0.0004; Fig. 3a). Despite no reduction in cell viability at earlier time points, the cell-cycle progression was already impaired after 48 h of treatment (10-7 M; Fig. 3b). At this time point, we observed increased percentage of cells in the G0/G1 phase (p = 0.008), at the expense of G2/M decline (p = 0.002) in calcitriol treated cells. Accordingly, we observed reduced mRNA levels of the G0/G1-S cell- cycle progression markers CCND1 (p = 0.0002), CDK4 (p = 0.001),
<0.0001
edge #
0.0011
50
1.5
>0.9999
Median M-values
1.0
9
0.5
808
0.0
30
-0.5
-1.0
Height
High
P
HighB
Low
20
A
54
B
53
10
51
52
48
47
50
M
49
A
43
$
39
40
30
D
41
42
S
30
PACT097_High
14
SE
&
2
31
N
N
N
2
S
8
P
20
0
.
PACTO82_Low
-
4
-
N
pACT001_High
pACT064_High
pACT073_High
pACT012_High
PACT101_High
H295R_unknown
pACT029_High
pACT050_High
pACT038_High
pACT036_High
pACT075_High
pACT035_High
pACT025_High
pACT030_High
pACT060_Low
PACTO96_Low
pACT003_Low
PACTO46_Low
PACTO37_Low
PACTO78_Low
pACT095_Low
PACT107_Low
PACTO45_Low
pACTO49_Low
PACT026_Low
PACTOS9_Low
pACT094_Low
pACTO48_Low
pACTOS3_Low
PACTO81_Low
PACTO88_Low
pACT102_Low
PACTO54_Low
PACTO87_Low
pACTO65_Low
PACTO70_Low
pACTO67_Low
pACTO98_Low
pACT092_Low
PACTO17_Low
pACT055_Low
pACTO14_Low
PACTO28_Low
PACT010_Low
PACTO13_Low
pACT021_Low
pACT006_Low
pACT061_Low
pACTO90_Low
pACT009_Low
PACTO40_Low
pACT068_Low
0
pACTO85_Low
pACT093_Low
pACTO20_Low
pACT058_Low
HIGH
LOW
(a)
(c)
ns
DAPİ
VDR
MERGED
ns
*
10
mRNA (2-44Ct)
Control
1
0.1
0.01
0.001
Calcitriol
o
0.0001
High A
High B
Low
Methylation group
Seocalcitol
(b)
Control
Calcitriol (10-7 M)
3
mRNA (2-44Ct)
*
63x
63x_Zoom_3x
2
+
1
(e)
CYP24A1
(f)
0
VDR
CYP27B1
20
Control
10 -7 M
Control
Seocalcitol (10-7 M)
10-
3.
**
(d)
Calcitriol (10-7 M)
mRNA (4Ct)
0
mRNA (2-44Ct)
2
*
+
+
-10-
+
VDR
54 kDa
-20
1
Fibrillarin
-30
37 kDa
-40
0
B-actin
42 kDa
Calcitriol
Seocalcitol
VDR
CYP27B1
Cyto Nuc Cyto Nuc
CCNE1 (p <0.0001), and CDK2 (p=0.0001) (Fig. 3c). After 96 h, the percentage of cells in the S phase was also reduced (p = 0.0003). The treatment of H295R cells with seocalcitol (10-7 M, 48 h) triggered comparable results, as it reduced cell viability (p < 0.0001; Fig. 3d) and downregulated the expression of the aforementioned cell-cycle con- trolling genes (Fig. 3e).
3.3. Effects of VDR activation in the Wnt/B-catenin signaling
Like most human ACC, the H295R cell line bears constitutive Wnt/B- catenin signaling. Since CCND1 is a bonafide B-catenin target gene, that was downregulated by VDR activation in this cell line, we evaluated whether the Wnt/B-catenin pathway could be a mediator of the effects observed in cell proliferation. The treatments with calcitriol or seo- calcitol (10-7 M; 48 h) resulted in a significant reduction of the expression of the B-Catenin coding gene CTNNB1 (p < 0.01), of the
oncogene MYC (p < 0.001) - another one of B-Catenin’s targets - and the pathway’s repressor DKK3 (p = 0.01) (Fig. 4a and b). In accordance, B-catenin nuclear accumulation was weakened at the same time point (Fig. 4c), despite no changes in protein levels after 72 h of treatment with calcitriol (Fig. 4d).
3.4. Effects of VDR activation in the mRNA expression profile of H295R ACC cells
In order to comprehensive elucidate the effects of VDR activation in the mRNA expression profile of H295R cells, we performed the tran- scriptome sequencing of control and cells treated with either calcitriol or seocalcitol (10-7 M, 48 h). Between-treatment comparisons identified a total of 137 and 163 DEGs with calcitriol and seocalcitol, respectively. No DEG was identified when comparing calcitriol and seocalcitol treatments (Supplementary Figs. 3a-c). Ninety-nine DEGs were common
(a)
(b)
Cell viability (% of control)
120-
p = 0.0004
Calcitriol (M)
0
100-
100-
I
10
-9
Cell population (%)
**
G2/M
I
**
25
19
27
24
S
80-
1
10 -8
80-
15
**
10-7
17
GO/G1
60-
17
14
10 -6
60-
Sub-G1
40-
40-
**
53
60
51
56
20-
20-
0
48h
96h
0
Control
Calcitriol
Control
Calcitriol
(10-7 M)
48h
96h
(c)
(d)
2.0
Control
Calcitriol (10-7 M)
120
p = 0.02
p < 0.0001
Seocalcitol (M)
100-
0
mRNA (2-44Ct)
Cell viability (% of control)
1.5
10 -9
**
80-
10 -8
10 -7
1.0-
60-
10
-6
40-
0.5-
+
20-
0.0
0
CCND1
CCNE1
CDK4
CDK2
48h
96h
(e)
2.0
*
Control *
Seocalcitol (10-7 M)
*
mRNA (2-44Ct)
1.5-
+
1.0
±
+
+
+
F
0.5-
0.0
CCND1
CCNE1
CDK4
CDK2
to both treatments (69 down- and 30 up-regulated), whereas 38 were calcitriol-specific (29 down- and 9 up-regulated), and 64 were seocalcitol-specific DEGs (29 down- and 35 up-regulated) (Fig. 5a). These gene-sets were hierarchically clustered and the heatmaps showing the treated-cells’ mRNA expression profiles are shown in Fig. 5b and c. Full information about the DEGs is available in the Supplementary Material.
Pathway analysis demonstrated that the most over-represented pathways were common to both calcitriol and seocalcitol treatments (Fig. 6). These pathways included steroid hormone biosynthesis, tran- scriptional misregulation in cancer, and Rap1 signaling pathway, among others (Supplementary Fig. 4). Gene ontology ORA of the encoded DEGs also demonstrated comparable over-represented terms triggered by either calcitriol or seocalcitol treatments (Supplementary Fig. 5). The most represented biological process terms represented hormone biosynthetic and metabolic processes, and regulation of hormone levels. In line, the most represented molecular function terms were mainly
involved with steroid hydroxylase activity, heme binding, and oxidase activity. The full lists of over-represented pathways and terms are available in the Supplementary Material.
3.5. In vivo study
3.5.1. Effects of seocalcitol on H295R ACC xenograft growth
After demonstrating VDR downregulation in human ACC, and the effects of its activation on tumor proliferation in vitro, we investigated whether the treatment with seocalcitol could impair adrenocortical tumorigenesis progression in vivo. In order to do that, we established an H295R xenograft model in female NSG mice, in which the tumor take rate was 100%. The xenografts were established 7 weeks after the sub- cutaneous inoculation. One animal in the seocalcitol group was excluded due to tumor hemorrhage and necrosis before the end of the treatment, and its data were disconsidered in the analysis. The final numbers of animals and xenografts were: 5 mice and 10 tumors in the
(a)
(b)
2.0
Control
Calcitriol (10-7 M)
2.0
Control
Seocalcitol (10-7 M)
1.5-
**
mRNA (2-44Ct)
1.5
**
mRNA (2-44Ct)
**
**
1.0-
HE
1.0-
+
+
0.5
0.5-
I
0.0
0.0
SRFP1
DKK3
AXIN1
AXIN2
CTNNB1
TCF-7
MYC
SRFP1
DKK3
AXIN1
AXIN2
CTNNB1
TCF-7
MYC
(c)
Control
Calcitriol
(d)
DAPI
Calcitriol (10-7 M)
+
+
M
B-Catenin
92 kDa
B-Catenin
Fibrillarin
37 kDa
B-actin
42 kDa
Cyto Nuc Cyto Nuc
Merged
control group, and 4 mice and 8 tumors in the seocalcitol group.
Once we started the treatments, the tumors from the animals in the control group rapidly grew, reaching a 2.8-fold higher volume right after two weeks (Fig. 7a). At this time point, the tumors from the seocalcitol treated mice were significantly smaller, and this pattern persisted throughout the experiment. At the end of the treatment, the mean tumor volume of the seocalcitol treated mice was half of the control’s (p = 0.01) and remained smaller (p = 0.03) after another week (Fig. 7a and b). The xenografts’ weight and volume were positively correlated (Fig. 7c).
3.5.2. Effects of seocalcitol on H295R ACC xenograft histology
The excised xenografts were involved by a thin capsule and a few extracapsular bulges were observed (Fig. 7b). There were no signs of tumor vascular invasion. Histological analysis of tumors from both groups demonstrated similar characteristics (Fig. 8): highly proliferative areas, as demonstrated by pleomorphism, frequent mitosis - including atypical ones - and few apoptotic bodies. However, tumors from the seocalcitol treated mice presented larger necrotic fields.
3.5.3. Systemic effects of seocalcitol in H295R ACC xenografted mice
We monitored the animals for possible complications of seocalcitol treatment throughout the experiment. Despite maintaining the initial body weight during the 7 weeks preceding the treatment, mice from both groups presented weight reduction between the first and third weeks of treatment, which was regained in the fourth week (Fig. 9a). The animals’ body weight was similar between the groups throughout the experiment, as was their corrected body weight at the end of the study (control: 23.2 ± 1 vs seocalcitol: 23.6 ± 1 g, p = 0.66).
Since hypercalcemia is a critical side effect of supraphysiological levels of vitamin D3, we monitored the mice’s serum calcium levels
(Fig. 9b). Right before the establishment of the treatments, both groups presented normal serum calcium levels (7.0-11.0 mg/dL). At the end of the experiment, seocalcitol treated mice calcium levels were higher than controls, but only marginally above reference levels (p = 0.05).
As tumor autonomous steroid secretion is an important comorbidity of ACT, we measured the animals’ testosterone and cortisol serum levels. Seocalcitol treated mice presented significant lower testosterone levels (p = 0.01; Fig. 9c) and, unlike control’s (2 ± 1.2 ng/dL), undetectable serum cortisol.
We also examined whether mice systemic VDR signaling was affected. The mRNA levels in the kidneys of seocalcitol treated mice were upregulated for Vdr (p = 0.03) and Cyp24a1 (p = 0.04), and downregulated for Cyp27b1 (p = 0.0009) (Fig. 9d), which demonstrated systemic VDR activation upon the treatment with the seocalcitol.
4. Discussion
We have recently demonstrated that VDR tumor hypermethylation is associated with VDR mRNA underexpression and unfavorable outcome in a large cohort of pediatric patients with ACT (Bueno et al., 2022). Since our clinical data evidenced that dysfunctional VDR contributes to adrenocortical tumorigenesis, in the present study, we present proof-of-principle data showing that VDR signaling restoring in H295R ACC cells results in antiproliferative and anti-steroidogenic effects, rendering this approach a potential therapeutic target, especially for tumors bearing Wnt/B-catenin activation.
We did not observe VDR signaling under basal conditions in H295R cells, which coincides with VDR underexpression in ACC. Hence, we investigated the VDR methylation profile, hypothesizing that high methylation associates with low VDR expression in these cells. By retrieving and comparing VDR DNA methylation and mRNA data from
(a)
Down-regulated
Up-regulated
Control vs calcitriol
Control vs seocalcitol
29
69
29
35
30
9
Control vs seocalcitol
Control vs calcitriol
(b)
Regulated Genes
(c)
Category
Regulated Genes
Category
CYP21A2
POMIZILOP CYP21A2 2
VWF
ENSCI00000254732 2
GOTS
CYP21A2 1
NTM
CAA
TRIB2
TUBB4A
GIPHI 57
ASB4
FGFR4
POGFRB
ETV4
SEPTINA
ENSQ 00000295238 2
MMMP24
TMEMBGA
GLUL
FOXNA
TAGUN
GADOGA
STAP2
HOXC12
HADE
RAPIGAP
CIQTNF3 AMACA
ENSQ00000294157.1
SOGBIC2
ENSQ00000299700 1
Centralized Category
Centralized Category
MOPL
Counting
Calcitriol
TSPANI8 TSPAN18
4
Control
Counting
Control
4
Seocalcitol
a
TMEMINEB
2
0
0000272410.5
2
SOX13 SOR13
-2
0
HONGH!
4
-2
ENSG00000277531.3
4
ABCBI
WWARB!
KALAN SEMANA
CABLES!
EFLİ
TMEM164
LC1343
TBKBP!
PSG4
LMOD!
TMEM37
MPND
ABTB2
BAINPZ
GOF15
NUPRI
CEBPD
OGREF!
BOKAB2
L
ENSG00000249072 1
AMPGEF PAPGEF4
KLK15
PAGEA 1
ENSG00000215859 9
500000028-4531.1
CYP3A5
TENM4
LARC30
COLIGAT
CPA2
ENSG0000020-4329 4
DYSF
IGFBPS CLON2
PLACI
NOXI
MOP
Ev9010000028-9831.1
LAMAAR
FLOTI
(a)
KEGG Pathways -All Regulated Genes
(b)
Common
Steroid hormone biosynthesis
CA4
GLUL
Transcriptional misregulation in cancer
Steroid hormone biosynthesis
Transcriptional misregulation in cancer
RAPGEF4
Rap1 signaling pathway
Rap1 signaling pathway
Prostate cancer
RAP1GAP
Prostate cancer
Nitrogen metabolism
Cortisol synthesis and secretion
Retinol metabolism
FGFR4
Nitrogen metabolism
Log2FC
1.0
Chemical carcinogenesis - DNA adducts
0.5
Melanoma
-log2(pvalue)
0.0
6
ID1
CYP11B1
-0.5
Glioma
5
-1.0
Complement and coagulation cascades
size
4
PDGFRB
CYP21A2
2
Focal adhesion
3
3
Regulation of actin cytoskeleton
5
Ovarian steroidogenesis
NUPR1
6
Endocrine and other factor-regulated calcium reabsorption
CYP3A5
Signaling pathways regulating pluripotency of stem cells
GADD45A
CYP3A4
PI3K-Akt signaling pathway
Cushing syndrome
Cell adhesion molecules
IGF1
CYP3A7
Drug metabolism - cytochrome P450
ETV5
2
4
IL
R2
HSD381
0
6
Gene Numbers
ETV4
(a)
(b)
Tumor volume
0
-
L
L
800
*
-
5
5
700
Control
*
0
U
600
Seocalcitol
5
-
500
*
UN
ON
mm3
*
400
*
&
6
300
-
4
-
@
200
6
6
100
5
8
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
Weeks after xenograft
(c)
0
mml1
2
3
0
mmll
2
3
1.0
Tumor weight (grams)
r = 0.88
95% CI: 0.64 to 0.94
Control
Seocalcitol
0.8
p < 0.0001
0.6
0.4
0.2
Control
Seocalcitol
0.0
0
500
1000
1500
Tumor volume (mm3)
H295R cells with our pACT, we observed high VDR methylation signa- ture in the cell line, since it clustered within the branch formed by the most hypermethylated ACC. Moreover, the cells’ mRNA levels grown
under basal conditions were far lower than their ACC methylation group-mates and most ACT. Corroborating with the effect of methyl- ation on VDR mRNA levels in adrenocortical tissues, Nanao et al. have
V
20 um
20 um
80 um
80 pm
(a)
(b)
Body weight
Serum calcium
30
13-
*
Control
28-
Seocalcitol
12-
*
26-
11-
grams
mg/dL
10-
24-
9-
22-
8.
20
7
20
6
0
0
0
1
2
3
4
5
6
7
8
9
10
11
1 12 1213
0
5
Weeks after xenograft
Weeks of treatment
(c)
(d)
Testosterone
Kidney mRNA
10
*
100
p = 0.01
*
75
1
ng/dL
2-44Ct (log10)
50
0.1
25
0
0.01
Control Seocalcitol
Vdr
Cyp24a1 Cyp27b1
. Control . Seocalcitol
recently demonstrated VDR promoter hypomethylation, increased levels of mRNA and protein expression in aldosterone producing adenomas (APAs) - a non-malignant type of ACT - when compared to non- functioning adrenocortical adenomas (NFAs). This finding was more preeminent in APAs harboring mutations in the gene coding the x1 subunit of the Na+/K + -ATPase (ATP1A1) (Nanao et al., 2022).
In mice, it is well established that autoregulation of Vdr mRNA by active vitamin D3 is mediated directly by several enhancers within the receptor itself (Zella et al., 2010; Lee et al., 2015). On the other hand, even though human VDR has a functionally conserved region analogous to a mouse highly active transcriptionally vitamin D3 response element (VDRE) (Zella et al., 2006), there is conflicting evidence about its ho- mologous up-regulation by calcitriol (Pan et al., 1991; Wiese et al., 1992; Marchwicka et al., 2016). In the present study, we not only observed a significant increase in VDR mRNA and protein levels, but also, the receptor shuttling into the cell nucleus upon treatment of human H295R cells with calcitriol and seocalcitol. Interestingly, it was previously demonstrated that VDR increment in human cells by calci- triol does not depend exclusively on its transcriptional activity, but also on posttranslational stabilization of the receptor itself and on increment of its half-life (Davoodi et al., 1995; Kongsbak et al., 2014). Our qPCR results demonstrated downstream VDR signaling, as shown by massive transactivation of its target gene CYP24A1 - the best evidence of VDR’s activation - and the repression of CYP27B1 - another vitamin D3 metabolizing enzyme, which is negatively regulated by VDR signaling. Of note, differently from other nuclear receptors, VDR can be found in some tissue-specific cell nucleus, even in the absence of ligands, where it controls target gene’s expression by switching between repressing and activating states depending on ligand availability (Long et al., 2015). However, this mechanism seems to be inactive H295R cells, since they lack nuclear VDR under basal conditions. The aforementioned obser- vations demonstrated that the treatment of H295R cells with vitamin D3 can surpass the effect of VDR hypermethylation and activate it’s signaling. Unlike H295R cells and similar to APAs, H295R-derived HAC15 ACC cells carrying an ATP1A1 mutation present increased VDR protein levels (Nanao et al., 2022). ATP1A1 mutation induces cell proliferation and tumorigenesis (Kobuke et al., 2021), probably due to increased intracellular Ca2+ uptake and/or acidification, as demon- strated in H295R cells (Stindl et al., 2015). However, the proliferation of HAC15 cells with mutant ATP1A1 is inhibited upon VDR suppression, and not upon upregulation by calcitriol supplementation (Nanao et al., 2022). These observations highlighting the importance of VDR signaling to proliferation and that it may vary according to different adrenocor- tical tissues.
VDR restoring in H295R cells with calcitriol and seocalcitol reduced cell viability. Similar to our results, and using much higher calcitriol concentrations than ours, Rubin et al. recently demonstrated inhibition of H295R cell proliferation in a dose and time-dependent manner, with an IC50 of 3 x 10-6 M for the same time of treatment (96 h) (Rubin et al., 2020). Moreover, the authors also demonstrated that calcitriol had an additive effect on subtherapeutic doses of mitotane over H295R cell proliferation, demonstrating a beneficial therapeutic effect of vitamin D3 as a co-adjuvant agent.
Since the role of VDR signaling in repressing B-catenin activity was well demonstrated in other types of cancers that progress with uncon- trolled Wnt/B-catenin signaling (Aguilera et al., 2007; Beildeck et al., 2009; Johnson et al., 2015; Muralidhar et al., 2019), at first, we decided to purposely investigate it on H295R ACC cells. In line, the repression of the Wnt/B-catenin pathway was also observed in our study. We demonstrated this effect by the reduction of B-catenin nuclear staining and mRNA levels, as well as its targets CCND1 and MYC, and deregu- lation of the repressors DKK3 and AXIN1 (qPCR). In accordance, Rubin’s study also demonstrated reduction of B-catenin mRNA and nuclear expression (Rubin et al., 2020). These data suggest that VDR antitumor effects in adrenocortical cells seem to be involved with the impairment of aberrant B-catenin activity.
The Wnt/B-catenin signaling controls cell proliferation, being the up- regulation of cell cycle progression one of the major mechanisms involved in ACT pathogenesis (Assié et al., 2014; Pinto et al., 2015; Szabó et al., 2010; Tömböl et al., 2009). We demonstrated that cell cycle arrestment was the underlying mechanism involved in H295R prolifer- ation decline, with no apparent induction to apoptosis. Besides CCND1, which is a B-catenin’s target, the mRNA levels of other cell cycle pro- gression markers were repressed. Cell cycle arrestment following depletion of B-catenin-dependent transcription by interfering RNA or pharmacological blockage was previously demonstrated in H295R cells, but both approaches also induced apoptosis (Doghman et al., 2008; Gaujoux et al., 2013; Leal et al., 2015; Salomon et al., 2015). In agree- ment with our results, the treatment of H295R cells with a 30-fold higher calcitriol concentration reduced cell proliferation via cell cycle arrest- ment without inducing apoptosis (Rubin et al., 2020). The moderated effect observed on cell proliferation and the absence of apoptosis prompted by calcitriol can be explained, at least in part, by the induction of CYP24A1 expression and, consequently, vitamin D3 degradation.
Aiming to comprehensively elucidate the signaling pathways acti- vated by VDR’s signaling, we also evaluated the transcriptome land- scape of calcitriol and seocalcitol treated cells. Taking advantage of this analysis, we found novel pathways to be investigated in order to clarify the mechanisms involved in the adrenocortical antitumor effects of VDR activation. We observed, as expected, that the most DEGs were common to both treatments, and so, were also the most over-represented path- ways and GO terms. Among them, steroid hormone biosynthesis pathway - as expected - and some pathways involved with cancer process were underscored. Of note, one of the most affected pathways was the Rap1, which has close relation with Wnt’s, being part of one of its noncanonical pathways: the Wnt/Rap1 pathway. In this context, upon Wnt-Frizzled initial signaling, Rap1 regulates actin cytoskeleton and/or cell adhesion during vertebrate gastrulation (Tsai et al., 2007). Interestingly, also using H295R ACC cells, Aumo et al., have previously demonstrated the importance of Rap1 as an effector of the exchange protein directly activated by 3’,5’-cyclic adenosine 5’-monophosphate (cAMP) 2 (EPAC2B), a sensor protein that mediates cAMP-induced ef- fects on cytoskeleton integrity and cell migration (Aumo et al., 2010). In line, we observed similar effects upon these cells in response to calcitriol treatment: morphology changes from flat and adherent spindle-to spherical-shaped cells.
The antiproliferative effects of synthetic vitamin D3 analogs have been demonstrated in several animal models of human cancers (Duffy et al., 2017). Seocalcitol - also known as EB1089 - is a structural analog of calcitriol, that has affinity to activate IP9-type VDREs. This affinity renders stable conformation of VDR-RXR-VDRE complexes and is correlated with potent antiproliferative/growth arrest effects, com- bined with reduced calcemic actions of seocalcitol (Nayeri et al., 1995; Quack and Carlberg, 1999). In vitro, seocalcitol had comparable effects to calcitriol in H295R ACC cells. Hence, we investigated whether its potential against ACC proliferation would translate in vivo. By devel- oping an H295R xenograft mouse model, we were able to recapture histological and hormonal features previously described by Logié et al. (2000). Upon treatment with seocalcitol, we observed a repression of tumor growth right after two weeks, resulting in a mean tumor volume ~50% smaller than control mice, throughout and after one week of the end of the treatment. In agreement with in vitro findings, the histological examination revealed that xenografts exposed to seocalcitol presented more extensive necrosis and lesser proliferative areas, with no apparent increment in apoptosis. In line, there is evidence about impaired apoptosis pACT from our cohort, since they have distinct profile of apoptosis-related genes than pediatric normal adrenals (Lorea et al., 2012). This suggests that antiapoptotic mechanisms might be involved in the biology and progression of ACT, and allow us to speculate that VDR impairment is involved with tumor growth, viability, and malig- nant progression permissiveness, and not necessarily with modulation of apoptosis in these tumors.
Tumor steroid secretion is associated with increased morbidity of ACT patients (Antonini et al., 2014; Else et al., 2014). In vitro, it has been demonstrated that H295R cells treated with different doses of calcitriol or PNU-74654, a B-catenin signaling inhibitor, reduced steroid secretion (Leal et al., 2015; Lundqvist et al., 2010; Pilon et al., 2014). Moreover, H295R xenografts autonomous steroid secretion are not only able to increase mice serum steroid levels, but also to yield non-murine steroids, like cortisol (Logié et al., 2000). In our study, seocalcitol treatment resulted in lower testosterone and undetectable cortisol levels in H295R xenografted female mice, which further validated our in vitro RNA-seq findings on steroid hormone biosynthesis and proved a beneficial ther- apeutic effect of the analog. Of note, the aforementioned effects were not accompanied neither by important hypercalcemia nor by weight loss, which are undesirable side effects of therapeutic doses of vitamin D3. This observation is valuable, since we fed the mice, a standard chow containing calcium and vitamin D3 throughout the experiment, in order to reflect the physiological effects of seocalcitol administration.
Physiologically, in order to antagonize VDR signaling, Cyp24a1 transcription is activated once vitamin D3 levels rise in its target tissues (Jones et al., 2012). The increment in Cyp24a1 mRNA, in addition to Cyp27b1 and Vdr modulation, was observed in the kidneys - vitamin D3-targets - of seocalcitol treated mice, which demonstrates the ana- log’s physiological bioavailability and reinforces systemic VDR activa- tion. These results demonstrate that calcitriol analogs can overcome the dose-limiting restriction of calcitriol therapeutic use. Moreover, in addition to antiproliferative effects, the activation of VDR signaling in ACT could also be beneficial by decreasing morbidity, especially in those patients experiencing severe consequences of prolonged exposure to steroids (Else et al., 2014). Further preclinical studies using clinically approved vitamin D3 analogs - such as those used for the treatment of secondary hyperparathyroidism, psoriasis and osteoporosis (Leyssens et al., 2014; DeLuca and Plum, 2016) - alone or in combination with mitotane, may validate the therapeutic effects of VDR signaling for the treatment of patients with ACT.
We recognize that the use of only one ACC cell line is not ideal and it can be considered a limitation of our study. Nevertheless, due to the features presented by H295R cell line, we believe that - even in spite of being adult derived - it is a reliable preclinical model for our study, since it: (i) has a mutational landscape which resembles aggressive ACC, including mutations in known driver-genes like ATRX and TP53, besides harboring the p.S45P, which confers them constitutive abnormal Wnt-B- catenin activation, a feature often observed in both pediatric and adult ACC (Leal et al., 2011; Mermejo et al., 2014; Tissier et al., 2005; Nic- olson et al., 2019); (ii) supports adrenal steroid secretion pattern, which enables the study of systemic effects of tumor autonomous hormone secretion (Rainey et al., 1994); (iii) presents High VDR methylation signature, which is associated with reduced VDR mRNA levels, higher tumor burden and unfavorable patient outcome (Bueno et al., 2022); (iv) presents impaired VDR expression/signaling, like observed in most ACC (Pilon et al., 2014; Bueno et al., 2022). We are aware of the recent advances in the generation of novel ACC preclinical models, like cell lines and xenograft models (Hantel et al., 2016; Kiseljak-Vassiliades et al., 2018; Landwehr et al., 2021; Pinto et al., 2013). The replication of our findings in these novel models may further undercover the mecha- nisms involved in VDR activation in different ACC genomic back- grounds, but to solely use them could mitigate or deviate the observation of the effects of vitamin D3 on adrenal tumorigenesis, since they harbor selected molecular features. However, to our knowledge, these models are not currently commercially available.
In conclusion, we demonstrated that, like most adult and pediatric ACC, H295R cells present high VDR methylation signature, which can be responsible for its underexpression and signaling inactivation under basal conditions. Moreover, our preclinical data support the importance of activating VDR signaling to promote antiproliferative effects in H295R xenografts, suggesting that active vitamin D3 analogs may be a potential therapeutic option for ACT, accruing a novel approach for the
clinical management of patients.
CRediT authorship contribution statement
Ana Carolina Bueno: Conceptualization, Methodology, Investiga- tion, Formal analysis, Writing, Writing - original draft. Candy Bellido More: Methodology, Investigation, Formal analysis. Junier Marrero- Gutiérrez: Methodology, Software, Formal analysis. Danillo C. de Almeida e Silva: Methodology, Software. Leticia Ferro Leal: Meth- odology, Writing, Writing - original draft. Ana Paula Montaldi: Methodology, and, Investigation. Fernando Silva Ramalho: Method- ology, Formal analysis. Ricardo Zorzetto Nicoliello Vêncio: Method- ology, Software, Formal analysis, Writing, Writing - original draft. Margaret de Castro: Resources, Writing, Writing - original draft, Funding acquisition. Sonir Roberto R. Antonini: Conceptualization, Formal analysis, Supervision, Resources, Writing, Writing - original draft, Funding acquisition.
Declaration of competing interest
The authors declare no conflicts of interest.
Data availability
Data will be made available on request.
Acknowledgements
We thank Professor Elza T. Sakamoto-Hojo (Faculty of Philosophy, Sciences and Letters at Ribeirao Preto) for the use of the Flow Cytometer, and Aline Turatti, PhD. (Laboratory of Dermatology; Molecular Biology, FMRP-USP) for technical support. We also thank Professor Dr Leandro M. Colli and Dr Fernanda B. Coeli-Lacchini for their support with mRNA sequencing. This work was funded by the Coordination for the Improvement of Higher Education Personnel (ACB) and the Sao Paulo Research Foundation (FAPESP) grants 14/03989-6 (MC and SRA), 15/ 19663-5 (SRA), 17/17737-7 (CCBM), 19/00860-6 (ACB), 21/04368-9 (DCAS), and 22/04883-3 (JMG).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi. org/10.1016/j.mce.2022.111757.
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