Abstract 3: OncomiR-155 targets oncogenes HDAC4 and BCL6 in a murine B cell leukemia model: A paradigm shift in the oncogenic mechanisms of microRNAs

Abstract

Abstract miR-155 belongs to a family of non-coding RNA, namely microRNAs (miRNAs) and is over-expressed in several lymphoid malignancies including chronic lymphocytic leukemia and diffuse large B-cell lymphomas. Transgenic over-expression of miR-155 in mouse B cells has been shown to induce pre-B cell proliferation followed by high-grade B-cell lymphoma. miR-155 exerts its oncogenic activity by targeting directly or indirectly tumor suppressor genes-SHIP-1 and C/EBPβ in B cells. To further characterize the mechanisms by which miR-155 contributes to leukemogenesis we did mRNA profiling of the B cells from the transgenic (TG) miR-155 mice spleens. Interestingly, we found that an oncogene Bcl-6 (B cell leukemia 6) was down regulated in the miR-155 TG B cells. Bcl-6 is a transcriptional repressor and a key regulator of germinal center B-cell development where antigen activated B cells differentiate to memory B cells or antibody producing plasma cells. Bcl6 is also involved in repression of cell cycle arrest and apoptosis associated genes by recruiting corepressors like histone deacetylases (HDACs). Further analysis of miR155 targets indicated another oncogene hdac4 to be a potential target. Luciferase reporter assay validated hdac4 as a direct target of miR155 which was further confirmed by 50% reduction in hdac4 mRNA and protein levels in TG mice by RT-PCR and immunoblotting respectively. To further investigate the effect of downegulation of hdac4 and Bcl6, we hypothesized that Bcl6 is functionally inactivated due to increased acetylation in TG mice and is targeted for ubiquitin mediated proteasome degradation due to downregulation of hdac4. Increased levels of acetylated Bcl6 in immunoprecipitated Bcl6 from total splenocytes of TG mice compared to WT control mice confirmed our hypothesis. This partly explains the post-translational regulation of Bcl6 indirectly by miR-155 through HDAC4. We further confirmed these findings in vitro in cultured cells lines with high and low expression of miR-155 and observed similar results of down regulation of HDAC4 and BCL6. In addition, we also observed significantly decreased proliferation of cells with high expression of miR-155 compared to cell lines with low expression of mir-155. mRNA analysis also showed significantly upregulation of genes involved in cell cycle arrest such as Cyclin D2/ccnd2, Cdkn1a and Inhibitor of differentiation 2/Id2) in TG mice compared to WT. These findings enlighten the complex interplay between tumor suppressor and oncogene pathways affected by a single gene, that when over-expressed results in leukemia. This indicates a shift in the paradigm of typical oncogenic miRNAs mechanisms, whereby they can act, not only by targeting tumor suppressors but also oncogenes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3.