MiR-155 Amplifies AKT and NFkB Signaling By Targeting Multiple Regulators of BCR Signal in DLBCL

Introduction

The B-cell receptor (BCR)- activated PI3K pathway plays a critical pro-survival function in normal B-lymphocytes and certain B-cell malignancies. Multiple mechanisms are involved in triggering and modulation of the BCR signal amplitude. MiR-155 has emerged as a positive regulator of PI3K signaling in multiple malignancies, including DLBCL, through targeting negative modulator of this pathway, SHIP-1. In the present study we have searched for new targets of miR-155, which might play a role in the deregulation of AKT and NFkB signaling in DLBCL.

Methods

MiR-155 target prediction was performed with PicTar, Miranda and TagetScan algorithms. Predicted miR-155 targets were validated with 3'UTR luciferase reporter assays in HEK293 cells. MiR-155 expression was modulated through transfection with miR-155 mimic or miR-155 inhibitor. The consequences of the miR-155 perturbations were assessed in DLBCL cell lines by proliferation assays (MTS) and immunoblotting with antibodies against predicted miR-155 targets and p-AKT. The DEPTOR silencing in DLBCL cells was achieved with retroviral shRNA vector and its consequences were assessed using proliferation assays and immunoblotting. DEPTOR mRNA expression and survival of DLBCL patients was determined using publicly available microarray data (Lenz et al, 2008, GEO accession GSE10846).

Results

Using miRNA target finding algorithms, we identified miR-155-matching sequences in 3'UTRs of two genes involved in SYK/PI3K/AKT pathway regulation: c-CBL (SYK ubiquitin E3 ligase) and DEPTOR (an mTOR phosphatase). MiR-155 suppressed the luciferase activities of vectors containing 3'UTR fragments from SHIP-1, c-CBL and DEPTOR genes with wild-type, but not mutant miR-155 seed sequence. To establish a link between miR-155 and c-CBL or DEPTOR, DLBCL cell lines were transfected with a control non-targeting miR or miR-155 mimic. The control miR did not affect the protein level of SHIP-1, c-CBL or DEPTOR, whereas introduction of miR-155 resulted in a decrease in expression of these proteins. The repression of miR-155 target genes was accompanied by increased phosphorylation of AKT. As expected, neutralization of endogenous miR-155 in U2932 cell line by anti-miR-155 exhibited opposite effects. In addition to modulation of SYK/PI3K/AKT pathway, miR-155 has been reported to modulate NFĸB activity. To determine whether miR-155 affects NFĸB in DLBCL, we assessed transcript abundance of the genes regulated by NFĸB (CD40, BFL-1, RelB, IĸBα, A20, MIR155HG) in U2932 cell line with reduced miR-155 level. Repression of endogenous miR-155 in these cells led to marked downregulation of NFĸB-controlled genes, indicating that miR-155 amplifies NFĸB signaling in this DLBCL cell line. Consistent with this, anti-miR-155 sensitized U2932 cells to ibrutinib.

Since the function of a newly identified miR-155 target, the mTOR phosphatase DEPTOR, in DLBCL has not been elucidated, we silenced the expression of this protein with shRNA. Attenuated protein level of DEPTOR enhanced activity of AKT and promoted proliferation of SU-DHL4 cell line. To determine whether expression level of DEPTOR correlates with patient survival, we analyzed data from gene expression studies. Higher DEPTOR mRNA level in primary DLBCL biopsies was associated with longer overall survival (log rank test, p=0.018). Collectively, these data suggest that DEPTOR plays a tumor suppressor function in DLBCL.

Conclusions

Our data underscore the role of miR-155 in the regulation of AKT and NFkB prosurvival signaling in DLBCL. MiR-155 regulates AKT signaling not only by decreasing expression of SHIP-1, but also by modulating the abundance of c-CBL and DEPTOR. DEPTOR modulates AKT activity and its silencing promotes proliferation of DLBCL cells, suggesting that DEPTOR functions as a tumor suppressor in DLBCL.