Study could lead to treatment for a type of cancer that affects children’s endocrine system

Experiments conducted at the University of São Paulo showed that expression of the gene that encodes the vitamin D receptor is greatly reduced in pediatric adrenocortical tumor cells. When the gene was activated, an anti-proliferation effect was observed. The only treatment currently available is surgical removal of the tumor.

By Maria Fernanda Ziegler | Agência FAPESP – Researchers at the University of São Paulo (USP) in Brazil have discovered a cellular mechanism associated with the formation of pediatric adrenocortical tumors, located in the adrenal glands on top of the kidneys.

Studies involving cultured cells and mice showed for the first time that activation of the gene that encodes the vitamin D receptor (VDR) inhibits the growth of this kind of tumor. The discovery could lead to the development of novel therapies for the disease. Currently, the only option is surgical removal of the tumor.

“We proved with these experiments that activation of the VDR gene blocks proliferation of tumor cells. We restored this pathway, which exists in healthy cells but tends to be inactive in tumor cells,” said Sonir Antonini, professor of pediatric endocrinology in the Department of Puericulture and Pediatrics at the Ribeirão Preto Medical School (FMRP-USP) and principal investigator for the studies.

The discovery resulted from three studies supported by FAPESP under the aegis of a Thematic Project led by Margaret de Castro, who is also a professor at FMRP-USP.

The results are reported in three articles published in the European Journal of Endocrinology, in Endocrine-Related Cancer, a journal of the Society for Endocrinology in the UK, and in Molecular and Cellular Endocrinology.

The first author of all three articles is nutritionist and pediatric endocrinologist Ana Carolina Bueno, who has a postdoctoral fellowship from FAPESP.

According to the researchers, pediatric adrenocortical tumors are rare, accounting for about 0.2% of all pediatric cancers, but they are up to 18 times more frequent in Brazil, especially in the South and Southeast. Because these tumors are located in components of the endocrine system responsible for producing hormones such as cortisol, aldosterone and androgens, most children with the disease present with symptoms of hormone imbalance such as precocious puberty or weight gain with stunted growth.

Methylation

The first part of the investigation consisted of an analysis of tumor cell DNA methylation, a biochemical modification in which a methyl group is added to the DNA molecule via the action of enzymes. DNA methylation is a natural process and one of several epigenetic mechanisms that cells use to control gene expression. When dysregulated, however, it can cause cells to malfunction and may contribute to the development or progression of cancer.

With the aid of bioinformatics tools, the researchers separated patient data into two groups with more and less aggressive tumors. “We were able to identify the methylation profile of patients with very aggressive tumors, corresponding to 20% of adrenocortical cancer cases. These tumors metastasize rapidly, causing more complications and deaths,” Antonini told Agência FAPESP.

The analysis showed that methylation was more intense in parts of tumor DNA than in healthy cells. “We extracted tumor DNA, submitted the genetic material to methylation analysis, and only then sequenced it in order to find out in which region the biochemical modification occurred. We analyzed a very large number of cases and were eventually able to identify a specific phenotype for these more severe patients,” Antonini said.

VDR gene

The methylation analysis also served as an important tool to explore novel genes and patterns linked to tumor formation. Based on the results of the first study, the researchers conducted in vitro experiments (in cells) and in vivo experiments (in mice) to investigate the role of the VDR gene in the formation of adrenocortical tumors.

“We observed a reduction in expression of the vitamin D pathway due to tumor tissue VDR gene hypermethylation, which doesn’t occur in normal tissue,” Antonini said.

The researchers demonstrated for the first time that expression of VDR is reduced in these tumors. “Above all in the most aggressive tumors [20% of cases], but not only in these, levels of the protein and messenger RNA both fall significantly, owing to VDR hypermethylation in tumor cells only,” he said.

The reduction in VDR expression was measured in tumor tissue samples from 108 pediatric patients treated at FMRP-USP’s general and teaching hospital (Hospital das Clínicas) and Boldrini Children’s Cancer Treatment Center in Campinas.

“The methylation profile showed that VDR was a possible candidate for more detailed study. It’s an important gene in other ways, too, with functions relating to control of growth and the cell cycle,” Antonini said.

Prior research by the group showed, for example, that the vitamin D pathway interacts with intracellular processes important to tumor formation, including the Wnt/beta-catenin signaling pathway. “Alterations in this pathway, such as when it functions too much, are among the main molecular abnormalities observed in pediatric tumors,” he said.

The researchers also conducted experiments on tumor cells and mice with adrenocortical tumors. “In both in vitro and in vivo trials, we found that tumor cells ceased to proliferate when we increased VDR expression. In other words, it had an anti-tumor effect,” he said, adding that the finding does not suggest the need for vitamin D supplementation in these cases. “What happens is a problem in the tumor tissue’s vitamin D pathway involving a loss or very substantial reduction in expression of VDR [the gene that encodes the protein that binds to vitamin D and enables it to act in the organism],” he said.

The article “Vitamin D receptor hypermethylation as a biomarker for pediatric adrenocortical tumors” is at: eje.bioscientifica.com/view/journals/eje/186/5/EJE-21-0879.xml. 

The article “DNA methylation is a comprehensive marker for pediatric adrenocortical tumors” is at: erc.bioscientifica.com/view/journals/erc/29/11/ERC-22-0145.xml.  

The article “Vitamin D receptor activation is a feasible therapeutic target to impair adrenocortical tumorigenesis” is at: www.sciencedirect.com/science/article/abs/pii/S0303720722002052?via%3Dihub.