MicroRNA Mediates DNA Demethylation Events Triggered by Retinoic Acid during Neuroblastoma Cell Differentiation

Abstract

Neuroblastoma is an often fatal pediatric cancer arising from precursor cells of the sympathetic nervous system. 13-Cis retinoic acid is included in the treatment regimen for patients with high-risk disease, and a similar derivative, all-trans-retinoic acid (ATRA), causes neuroblastoma cell lines to undergo differentiation. The molecular signaling pathways involved with ATRA-induced differentiation are complex, and the role that DNA methylation changes might play are unknown. The purpose of this study was to evaluate the genome-wide effects of ATRA on DNA methylation using methylated DNA immunoprecipitation applied to microarrays representing all known promoter and CpG islands. Four hundred and two gene promoters became demethylated, whereas 88 were hypermethylated post-ATRA. mRNA expression microarrays revealed that 82 of the demethylated genes were overexpressed by >2-fold, whereas 13 of the hypermethylated genes were underexpressed. Gene ontology analysis indicated that demethylated and re-expressed genes were enriched for signal transduction pathways, including NOS1, which is required for neural cell differentiation. As a potential mechanism for the DNA methylation changes, we show the downregulation of methyltransferases, DNMT1 and DNMT3B, along with the upregulation of endogenous microRNAs targeting them. Ectopic overexpression of miR-152, targeting DNMT1, also negatively affected cell invasiveness and anchorage-independent growth, contributing in part to the differentiated phenotype. We conclude that functionally important, miRNA-mediated DNA demethylation changes contribute to the process of ATRA-induced differentiation resulting in the activation of NOS1, a critical determinant of neural cell differentiation. Our findings illustrate the plasticity and dynamic nature of the epigenome during cancer cell differentiation. Cancer Res; 70(20); 7874-81. ©2010 AACR.

Retinoic acid signaling and neuronal differentiation

“…miRNA profiles can predict survival of patients with neuroblastoma, therefore, making targeting miRNAs with antagomirs (oligonucleotides that block miRNA activity) a promising therapy [221]. In addition to altering miRNA expression, RA can induce genome-wide changes in DNA methylation by increasing expression of DNA methyltransferases and concomitant hypomethylation of promoters during neuroblastoma differentiation [222]. These changes in miRNA and DNA methylation alter the epigenetic landscape and ultimately affect the expression of oncogenes and tumorsuppressor genes.…”

N-myc and Noncoding RNAs in Neuroblastoma

“…All but 7 miRNAs were changed in the same direction when high-N-myc and low-N-myc states were compared in both species. Principal component analysis (PCA) suggested that N-myc regulates similar miRNAs in human and mice, including miRNAs with established functions in neuroblastoma, like members of the mir-17-92 cluster (54, 63), mir-9 (57), and mir-152 (78). The miRNA profiles across murine tumors were more homogeneous than in human tumors, which might reflect the inbred genetic environment in mouse versus human tumors (77).…”

“…Overexpression of mir-101 and let-7e in MYCNamplified neuroblastoma cells diminished N-myc levels, and reduced proliferation and clonogenic growth. While mir-101 is generally sparsely expressed in neuroblastoma and has established tumor-suppressor functions in other cancers [(100) and references therein], let-7 family members are upregulated during neuroblastoma cell differentiation (47,56,78,(101)(102)(103), suggesting tumor suppressor functions for these miRNAs in neuroblastoma.…”

The role of vitamins and minerals in modulating the expression of microRNA

“…Overexpression of miR-152 in SK-N-BE cells mimicked some, but not all, of the features of differentiation seen with retinoic acid treatment, suggesting that miR-152 is only partially responsible for phenotype in this system (118) .…”

“…Vitamin D supplementation is now being investigated in the treatment and prevention of these diseases, with a particular focus on its potential as a cancer chemopreventative, due to the identified role of the vitamin D receptor (VDR) in cell-cycle regulation and differentiation (86) . The active vitamin D metabolite, calcitriol (1,25-dihydroxyvitamin D 3 ), has long been known to directly (50) Choline deficiency (114) , methyl group deficiency (112) 126 (49) Vitamin C deficiency (131) 152 (118) Retinoic acid treatment (118) DNMT3 29 (47,48) Folate/methyl group deficiency (108) , retinoic acid treatment (123) 222 (95) Vitamin D treatment (90) Histone methyltransferase SUV39H1 125b (57) Vitamin E deficiency (142) , retinoic acid treatment (117,120) , vitamin D treatment (89) HDAC and HAT 206 (64) Retinoic acid treatment (117,120) 22 (94) Vitamin D treatment (92) HDAC 627 (96) Vitamin D treatment (96) DNMT, DNA-methyl transferase; HDAC, histone deacetylase; HAT, histone acetyltransferase.…”