Abstract 1040: A truncated DNMT3B protein (DNMT3B7) drives DNA methylation and suppresses growth in human neuroblastoma

Abstract

Abstract Cancer cells have an altered distribution of DNA methylation relative to normal cells. We have shown previously that human cancer cells express aberrant DNMT3B transcripts via abnormal gene splicing. Many of these aberrant transcripts encode truncated proteins, including DNMT3B7, one of the most commonly expressed aberrant isoforms expressed in cancer. In vitro, DNMT3B7 expression modifies the pattern of DNA methylation and gene expression. Neuroblastoma is a pediatric tumor of the neural crest cell lineage. A clinically aggressive disease and poor outcome is associated with CpG island methylator phenotype in neuroblastoma. We found that the expression of four or more DNMT3B transcripts, in primary neuroblastic tumors correlated with high-risk and stage IV disease. Further, the expression of ΔDNMT3B5 also correlated to high-risk and stage IV tumors. Additionally, higher levels of DNMT3B7 expression were detected in more differentiated neuroblastic tumors (ganglioneuroblastomas) compared to undifferentiated neuroblastomas. To determine if DNMT3B7 expression might alter neuroblastoma phenotype, we induced exogenous expression of DNMT3B7 in SMS-KCNR and LA1-55n cells, both aggressive neuroblastoma cell lines. Forced DNMT3B7 expression significantly inhibited neuroblastoma tumor growth in both cell lines. Histologic evaluation of the DNMT3B7-positive xenografts revealed decreased neuroblastoma cell proliferation, increased apoptosis, and inhibited angiogenesis. RNA-Sequencing of the DNMT3B7-expressing LA1-55n cells revealed dramatic decreased expression of FOS and JUN family members (FOS, FOSB, FOSL1, FOSL2, JUN, JUNB, JUND), which encode the components of the AP1 complex. In addition to acting as a transcription factor, AP1 can also antagonize the activity of retinoic acid receptors. We therefore hypothesized that with reduced levels of AP1, retinoic acid receptors would be more active and may drive the DNMT3B7-expressing cells toward differentiation. Among 24 genes that have been used as markers for differentiation in neuroblastoma, 19 have expression changes in DNMT3B7-expressing LA1-55n cells relative to control cells that correlate with a more differentiated phenotype. We also found higher levels of total genomic methylation in neuroblastoma cells with forced DNMT3B7 expression compared to controls in addition to gene promoter methylation changes that correlated to gene expression data. Our results demonstrate that high levels of DNMT3B7 increases total DNA methylation and inhibits neuroblastoma cell proliferation, angiogenesis, and tumor growth. Further knowledge regarding mechanisms by which DNMT3B7 regulates gene methylation may lead ultimately to the development of therapeutic strategies that reverse the epigenetic aberrations that drive neuroblastoma pathogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1040. doi:1538-7445.AM2012-1040