2.-PREMIOS de la Sociedad Latinoamericana de Endocrinología Pediátricia (SLEP) otorgados en el XXVI Congreso de la Sociedad Latinoamericana de Endocrinología Pediátricia, Buenos Aires, 2016.

SEGUNDO PREMIO

Vitamin D Receptor (VDR) is Underexpressed in Pediatric Adrenocortical Tumors and 1,25(OH)2D3/VDR Plays Antiproliferative Effects Via Suppression of Beta-Catenin and Cell Cycle Arrest in Adrenal Cells. Bueno, AC⁽¹⁾; Leal, LF⁽¹⁾; Gomes, DC⁽²⁾; Montaldi, AP⁽³⁾; Brandalise, SR⁽⁴⁾; Masterallo, MJ⁽⁴⁾; Cardinalli, IA⁽⁴⁾; Yunes, JA⁽⁴⁾; Martinelli Jr, CE⁽¹⁾; Tone, LG⁽¹⁾; Scrideli, CA⁽¹⁾; Moreira, AC⁽⁵⁾; Molina, CA⁽⁶⁾; Ramalho, F⁽⁷⁾; Ramalho, LNZ⁽⁷⁾; Tucci Jr, S⁽⁶⁾; Castro, M⁽⁵⁾; Antonini, SR⁽¹⁾

(1)Pediatrics, Ribeirao Preto Medical School, University of Sao Paulo. Ribeirao Preto, Brazil (2)Pediatrics, Federal University of Uberlandia. Uberlandia, Brazil (3)Genetics, Ribeirao Preto Medical School, University of Sao Paulo. Ribeirao Preto, Brazil (4)Boldrini Children’s Center. Campinas, Brazil (5)Internal Medicine, Ribeirao Preto Medical School, University of Sao Paulo. Ribeirao Preto, Brazil (6)Surgery and Anatomy, Ribeirao Preto Medical School, University of Sao Paulo. Ribeirao Preto, Brazil (7)Pathology, Ribeirao Preto Medical School, University of Sao Paulo. Ribeirao Preto, Brazil

ABSTRACT

Vitamin D Receptor (VDR) Is Underexpressed inPediatric Adrenocortical Tumors and 1,25(OH)2D3/ VDR Plays Antiproliferative Effects via Suppression of Beta-Catenin and Cell Cycle Arrest in Adrenal Cells Bueno, A.C.1; Leal, L.F.1; Gomes, D.C.2; Montaldi, A.P.3; Brandalise, S.R.4; Masterallo, M.J.4; Cardinalli, I.A.4; Yunes, J.A.4; Martinelli Jr, C.E.1; Tone, L.G.1; Scrideli, C.A.1; Moreira, A.C.5; Molina, C.A.6; Ramalho, F.7; Ramalho, L.N.Z.7; Tucci Jr, S.6; Castro, M.5; Antonini, S.R.1

1Pediatrics, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil; 2Pediatrics, Federal University of Uberlandia, Uberlandia, Brazil; 3Genetics, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil; 4Boldrini Children’s Center, Campinas, Brazil; 5Internal Medicine, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil; 6Surgery and Anatomy, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil; 7Pathology, Ribeirao Preto Medical School, University of Sao  Paulo, RibeiraoPreto, Brazil

Wnt/Beta-catenin pathway is activated in pediatric adrenocortical tumors (pACTs). The vitamin D3 (calcitriol-1,25(OH)2D3) receptor (VDR) was shown to be underexpressed in adult adrenocortical carcinomas (ACCs). It is possible that 1,25(OH)2D3/VDR interacts with beta-catenin in adrenal cells.

Aim: To investigate the role of 1,25(OH)2D3/VDR in pACTs tumorigenesis and its interaction with beta-catenin and adrenocortical cell proliferation.

Methods: Clinicopathological features and VDR expression were evaluated in 72 pACTs, 33 fetal (FAs) and 12 pediatric adrenals (pNAs) by qPCR and immunohistochemistry. In vitro, we evaluated in NCI-H295 cells the effects of VDR activation by 1,25(OH)2D3 (10–7 M) or inhibition (siRNA knock-down) on vitamin D pathway (CYP27A1 and CYP24A1), beta-catenin, and cell cycle markers (CTNNB1, MYC, CCND1, CDK4, CCNE1, and CDK2) mRNA expression (qPCR); protein expression (western blot), cell localization (immunofluorescence-IF), cell cycle (flow cytometry) and cell viability (MTS).

Results: VDR expression was observed mainly in the nucleus of FAs subcapsular cells (20th week) and progressively increased, extended to the cytoplasm and spread throughout the cortex in late gestation and postnatal adrenals. In pACTs, VDR staining was absent in 4.3%, nuclear/cytoplasmatic in 28.3% and cytoplasmatic in 67.4%. Strong VDR staining inversely associated with Weiss score (p < 0.001). Compared to pNAs, VDR mRNA expression was de creased in pACTs (p = 0.01), especially in carcinomas (p < 0.05).

In vitro, the activation of VDR by 1,25(OH)2D3 (48 h) was confirmed by decreased CYP27A1 (p  < 0.0001) and increased CYP24A1 (p < 0.001), VDR expression (mRNA: p = 0.001; protein: 87%) and nuclear cell staining. 1,25(OH)2D3/VDR activation arrested cell cycle in GO/G1 (53 to 60%; p < 0.01), decreased G2 (25 to 19%; p  < 0.05), and reduced the mRNA expression of G1-S markers CCND1 (p < 0.0001), CDK4 (p = 0.001), CCNE1 (p <0.0001), CDK2 (p = 0.001). MYC (p < 0.001) and CTNNB1 (p <0.001) mRNA expression were also reduced and beta-catenin staining (IF) was impaired. Cell viability was reduced after 96 h of 1,25(OH)2D3 treatment (–8.7%; p  < 0.001). VDR knockdown (–77%; 48 h) increased beta-catenin expression (79.5%; p = 0.01).

When activated by 1,25(OH)2D3, remaining VDR (23%) reduced MYC (–23%; p = 0.01) and CCND1 (–28%; p = 0.01) expression.

Conclusions: VDR plays a role in adrenocortical differentiation and is underexpressed in pACTs, mainly in pACC. In vitro, 1,25(OH)2D3/VDR inhibits beta-catenin, reducing adrenal cell proliferation and may emerge as a new antitumor target.