MicroRNA-155 Modulates Transforming Growth Factor-β Signaling In Chronic Lymphocytic Leukemia through Targeting of Casein Kinase γ Isoform 2

Abstract 3584

Chronic lymphocytic leukemia (CLL) is typically characterized by defects in programmed cell death rather than alterations in cell cycle regulation. Transforming growth factor β (TGFβ), a ubiquitously expressed growth factor, regulates multiple normal cellular responses including proliferation, differentiation, migration and apoptosis. Loss of growth inhibition by TGFβ is thought to contribute to the development and progression of a variety of tumors including CLL (DeCoteau et al., PNAS 1997). Approximately 40% of patients contain mutations in the signal sequence of TGFβ receptor 1 (TBR-1) in the form of substitutions or deletions (Schiemann et al., Cancer Detect Prev 2004). In the wild type form, the signal sequence contains a nine alanine stretch, which if truncated has been shown to impair signaling through the receptor and specifically, a truncated, six alanine form is associated with increased cancer risk (Pasche et al., Cancer Res 1999). TGFβ signaling can regulate expression of micoRNAs (miRNA), which are ~22 nucleotide-long RNA gene regulators. Deregulated miRNA expression has been implicated in tumorigenesis, including CLL. Several miRNAs have been shown to be over-expressed in CLL as compared to normal B cells (Fulci et al., Blood 2007). This includes miR-155, which is part of a 13-miRNA signature that has prognostic implications, including a shorter need-for-treatment interval (Calin et al., N Engl J Med 2005). Interestingly, miR-155 has been shown to be upregulated by TGFβ in murine mammary gland cells (Kong et al., Mol Cell Biol 2008). The goals of our study are to investigate the link between TGFβ signaling and miR-155 in CLL and to determine how the interaction between the two may contribute to the pathogenesis of CLL. Here we show that miR-155 is in fact upregulated by TGFβ in mouse splenic B cells and in human peripheral blood B cells. In CLL, miR-155 expression inversely correlates with the proportion of CLL cells harboring signal sequence mutation in TBR-1, consistent with miR155 regulation by TGFβ in vivo. To understand the role of TGFβ-induced miR-155 in CLL pathobiology, identification of specific target genes in the context of this disease is essential. To this end, we compared the gene (cDNA) expression profile between CLL with high miR-155 vs. low miR-155 expression and identified putative miR-155 target genes by selecting those genes that are differentially expressed in SAM analysis with lower expression in the high miR-155 group, and which harbor predicted miR-155 binding sites in their 3’ untranslated region (UTR). Based on this algorithm, we have identified casein kinase 1 gamma 2 (CSK1γ2) as a target for miR155 in CLL. CSK1γ2 is a negative modulator of the TGFβ signaling pathway by targeting the phosphorylated form of SMAD3 for degradation (Guo et al., Oncogene 2008). MiR-155 represses luciferase reporter gene expression by specific binding to the miR-155 site in the CSK1γ2 3’UTR. In addition, we found that CSK1γ2 itself is upregulated in B cells upon TGFβ stimulation, and treatment of human B cells with PNA miR-155 inhibitor (Fabani et al., Nucleic Acids Research 2010) further increases CSK1γ2 mRNA levels. Surprisingly, comparison of CSK1γ2 protein levels between CLLs with high or low miR-155 by Western blotting revealed higher CSK1γ2 protein expression despite lower CSK1γ2 mRNA levels, suggesting that miR-155 may enhance CSK1γ2 translation in CLL cells and implying an intriguing regulatory interaction between miR-155 and CSK1γ2. In summary, our data indicates that the variation of miR-155 seen in CLL is primarily a function of TGFβ signaling activity. Moreover, miR-155 is an important player in a complex auto-regulatory network in TGFβ signaling by fine-tuning the negative feedback mechanism on TGFβ signaling mediated by CSK1γ2. In CLL cells harboring TBR-1 with wild-type signal sequence, higher miR-155 levels may help modulate the TGFβ signaling activity to a level optimal for the survival or other pathobiological functions of CLL. Furthermore, since CLL cells are predominantly non-proliferating, our findings that miR-155 may enhance translation of CSK1γ2 provide support to the model of cell cycle dependence of microRNA functions (Vasudevan et al., Cell Cycle 2008).