Underexpression of the Vitamin D Receptor in Pediatric Adrenocortical Tumors and the Inhibitory Effect of 1α,25-Dihydroxyvitamin D3 on Beta-Catenin Signaling in Adrenocortical Tumor Cells

Presentation Number: MON 398
Date of Presentation: April 3rd, 2017

Ana Carolina Bueno*1, Leticia F. Leal2, Debora Cristiane Gomes3, Silvia R. Brandalise4, Maria José Mastellaro4, Izilda Aparecida Cardinalli4, José A. Yunes4, Carlos Eduardo Martinelli Jr.1, Luiz G. Tone1, Carlos A. Scrideli1, Ayrton C. Moreira1, Carlos Augusto F. Molina1, Leandra N. Z. Ramalho1, Fernando S. Ramalho5, Silvio Tucci5, Margaret de Castro1 and Sonir R. Antonini1
1Ribeirao Preto Medical School - University of Sao Paulo, Ribeirao Preto, Brazil, 2Barretos Cancer Hospital, Barretos, 3School of Medicine of Federal University of Uberlandia, Uberlandia MG, Brazil, 4Boldrini Children's Center, Campinas, Brazil, 5Ribeirao Preto Medical School, University of Sao Paulo

Abstract

The Wnt/Beta-catenin pathway activation is a hallmark of adrenocortical tumors (ACT). The vitamin D3 receptor (VDR) was shown underexpressed in adult adrenocortical carcinomas. It is possible that VDR and beta-catenin pathways interact in adrenocortical cells.

AIM: We investigated the role of 1,25-dihydroxydevitamin D3 (VitD3)/VDR signaling in the ACT tumorigenesis and its interaction with beta-catenin and adrenocortical cell proliferation.

METHODS: Clinicopathological features and VDR expression were evaluated in 72 pediatric ACTs, 33 fetal and 12 pediatric adrenals (NA) by qPCR and immunohistochemistry. In vitro, we evaluated in NCI-H295 cells the effects of VDR activation by VitD3 (10-7M) or inhibition (siRNA) on VitD3 metabolism (CYP24A1), beta-catenin and cell cycle markers (CTNNB1, MYC, CCND1, CDK4, CCNE1, and CDK2), DKK3 mRNA expression (qPCR), protein expression/localization (western blot and immunofluorescence-IF), cell cycle (flow cytometry), and cell viability (MTS). The TCF/beta-catenin complex was inhibited with PNU-74654 (10-4M) and the VDR signaling was evaluated (qPCR and IF).

RESULTS: VDR expression was observed in the nucleus of fetal adrenal subcapsular cells (20th week). It progressively increased, extended to the cytoplasm and spread throughout the cortex in late gestation and postnatal adrenals. In ACT, VDR staining was mostly cytoplasmic in 67.4%. Strong VDR staining inversely associated with Weiss score (p<0.001). Compared to NAs, VDR mRNA expression was decreased in ACT (p=0.01), especially in carcinomas (p<0.05). In vitro, VitD3 treatment (48h) increased CYP24A1 (p<0.001), VDR expression (mRNA: p=0.001; protein: 87%) and nuclear cell staining. VitD3/VDR activation arrested adrenocortical cells in the GO/G1 phase of the cell cycle (53 to 60%;p<0.01) and reduced the expression of G1-S transition markers CCND1, CDK4, CCNE1 and CDK2 (all p≤0.001). The oncogenic MYC (p<0.001) and CTNNB1 (p<0.001) expression were also reduced and beta-catenin staining (IF) was impaired. Cell viability was reduced after 96h of treatment (-8.7%;p<0.001). VDR knockdown (-77%;48h) increased beta-catenin expression (79.5%;p=0.01) both in cell nucleus and cytoplasm (IF). Post silencing treatment with VitD3 partially resettled VDR gene and nuclear expression (+40%p=0.09), and reduced MYC (p=0.01) and CCND1 (p=0.01) mRNA. PNU-74654 (48h) increased VDR gene (p=0.002) and nuclear expression, as well DKK3 mRNA (p=0.04).

CONCLUSION: VDR plays a role in adrenocortical cells differentiation and maintenance. In pediatric ACT, mainly in carcinomas, VDR was significantly underexpressed. VitD3/VDR and Wnt/beta-catenin pathways interacted in a way that VDR repressed beta-catenin and reduced adrenocortical cell proliferation, whereas TCF/beta-catenin inhibition increased VDR signaling. VDR activation may emerge as a new antitumor therapy for ACT.

 

Nothing to Disclose: ACB, LFL, DCG, SRB, MJM, IAC, JAY, CEM Jr., LGT, CAS, ACM, CAFM, LNZR, FSR, ST, MDC, SRA