MicroRNA miR-155 Inhibits Bone Morphogenetic Protein (BMP) Signaling and BMP-Mediated Epstein-Barr Virus Reactivation

MicroRNA miR-155 is expressed at elevated levels in human cancers including cancers of the lung, breast, colon, and a subset of lymphoid malignancies. In B cells, miR-155 is induced by the oncogenic latency gene expression program of the human herpesvirus Epstein-Barr virus (EBV). Two other oncogenic herpesviruses, Kaposi''s sarcoma-associated herpesvirus and Marek''s disease virus, encode functional homologues of miR-155, suggesting a role for this microRNA in the biology and pathogenesis of these viruses. Bone morphogenetic protein (BMP) signaling is involved in an array of cellular processes, including differentiation, growth inhibition, and senescence, through context-dependent interactions with multiple signaling pathways. Alteration of this pathway contributes to a number of disease states including cancer. Here, we show that miR-155 targets the 3′ untranslated region of multiple components of the BMP signaling cascade, including SMAD1, SMAD5, HIVEP2, CEBPB, RUNX2, and MYO10. Targeting of these mediators results in the inhibition of BMP2-, BMP6-, and BMP7-induced ID3 expression as well as BMP-mediated EBV reactivation in the EBV-positive B-cell line, Mutu I. Further, miR-155 inhibits SMAD1 and SMAD5 expression in the lung epithelial cell line A549, it inhibits BMP-mediated induction of the cyclin-dependent kinase inhibitor p21, and it reverses BMP-mediated cell growth inhibition. These results suggest a role for miR-155 in controlling BMP-mediated cellular processes, in regulating BMP-induced EBV reactivation, and in the inhibition of antitumor effects of BMP signaling in normal and virus-infected cells.Despite the limited genetic content of microRNAs, their pervasive role in controlling normal and pathology-associated cellular processes has become firmly established in recent years. The importance of microRNA dysregulation in cancer is well appreciated, and a number of oncomirs and tumor suppressor microRNAs have been identified (15). As a member of the oncomir class of microRNAs, miR-155 is implicated in lymphomagenesis and a wide array of nonlymphoid tumors including breast, colon, and lung (7, 16, 24, 39, 42, 43). Despite strong evidence implicating miR-155 in cancer etiology, the mechanisms through which miR-155 supports the tumor phenotype are unclear, possibly due to limited knowledge of how predicted targets may be involved in the phenotypic properties of cancer. On the other hand, miR-155''s roles in normal immune cell development and the adaptive immune response are much better understood (33, 41). These studies have demonstrated a critical role for miR-155 in immune cell activation and maturation. This evidence and other work (8, 40) have identified critical miR-155 targets whose downregulation is required for these processes.The Epstein-Barr virus (EBV) is a human DNA tumor virus that contributes to lymphoid and epithelial cell malignancies. As a herpesvirus, a unique aspect of the EBV infection cycle is the ability to exist in either a lytic replicative state or in a latent state in which no virus is produced. Depending in part on cell background, EBV utilizes multiple forms of latency gene expression programs. True latency and type I latency are defined by the expression of no protein coding genes or by expression of the episomal replication factor EBNA1 only. Type II latency is defined by the expression of EBNA1 and the latent membrane proteins, LMP1 and/or LMP2, and is the predominant form observed in epithelial tissues. Type III latency refers to expression of the full repertoire of latency genes, which are highly tumorigenic and are capable of growth-transforming naïve resting B cells. While this form of latency is not well tolerated in immunocompetent individuals except during early stages of infection (prior to the development of adaptive immunity to these proteins), type III latency-associated lymphoid malignancies are common in immunocompromised individuals. Expression of type III latency genes in B cells mimics antigen-dependent B-cell activation, and accompanying this activation is a substantial induction of miR-155 expression (17, 20, 23, 29, 44). While it is reasonable to assume that induction of miR-155 by the type III latency program plays a role in EBV-mediated B-cell activation and oncogenesis, little is known regarding the role of miR-155 in the virus life cycle or its tumor-promoting activities.Originally identified as cytokines critically involved in the regulation of osteogenic differentiation, bone morphogenetic proteins (BMPs) are now appreciated as having critical functions in a vast number of developmental processes. Dysregulation of BMP signaling is also implicated in disease states including cancer (1). The canonical signaling pathway stimulated by BMP receptor engagement is the phosphorylation of the SMADs (mothers against decapentaplegic homologs), SMAD1, SMAD5, and SMAD9, which facilitates active transport of these mediators from the cytoplasm to the nucleus, where they bind and activate cellular promoters. While these signaling mediators are considered to have fairly redundant activities, the influence of BMP activation can have widely distinct outcomes on a particular cell depending on cellular context (3, 27). These distinctions arise from the innate low-affinity DNA binding properties of SMADs and the concordant requirement for any of a broad range of cofactors that facilitate high-affinity binding to specific sets of promoters. Using this signaling mechanism, the phenotypic outcome of BMP receptor engagement is controlled by the level of activation of other signaling pathways and SMAD binding cofactors. While activation of BMP signaling appears to contribute to some cancer types, it inhibits other cancer types by promoting growth arrest and differentiation and by inducing senescence (1). In immune cells, BMP signaling has been shown by multiple groups to inhibit lymphocyte activation, maturation, and growth (2, 6, 13, 18, 19, 37). Here, we show that miR-155 inhibits BMP signaling by targeting multiple factors in the BMP signal transduction cascade. This function may be important during immune cell activation by preventing BMP from impeding this process, it may be important for the survival of EBV type III latency associated tumors by preventing BMP-mediated viral reactivation and cell death, and it may be relevant to other cancer types by blocking growth arrest properties of BMPs.