Downregulation of Deptor By MiR-155 Promotes Cell Survival through Activation of PI3K/AKT and NFkB Signaling in ABC-Type Diffuse Large B-Cell Lymphomas

Introduction

MiR-155 expression in DLBCLs is induced by NFkB and directly targets key regulators of B-cell maturation, motility and BCR signaling. In this study, we searched for new targets of miR-155 potentially involved in deregulation of BCR-PI3K/AKT and NFkB signaling in DLBCLs. We identified two new miR-155 targets: c-CBL (SYK ubiquitin E3 ligase) and DEPTOR (mTOR phosphatase). DEPTOR suppresses mTOR activity and in different tumors plays a role of either a tumor suppressor or an oncogene. Since the role of DEPTOR in DLCBLs cells has not been determined, we assessed the consequences of its inhibition in this malignancy.

Methods

Predicted miR-155 targets were validated with 3'UTR luciferase reporter assays. MiR-155 expression was modulated through transfection with miR-155 mimic or miR-155 inhibitor. The DEPTOR silencing in DLBCL cell was achieved with retroviral shRNA vector. DEPTOR mRNA expression and survival of DLBCL patients was determined using publicly available microarray data (Lenz et al, 2008, GEO accession GSE10846). Immunohistochemical assessment of DEPTOR expression was performed in 76 newly diagnosed DLBCL patients with available GCB/non-GCB designations based on Hans algorithm.

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 and DEPTOR. MiR-155 suppressed luciferase activities of vectors containing 3'UTR fragments from 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. Introduction of miR-155 resulted in decreased expression of c-CBL and DEPTOR, accompanied by a marked increase in phospho-AKT level. Inhibition of endogenous miR-155 in U2932 cell line by anti-miR-155 exhibited opposite effects. Using publicly available gene expression data (Lenz et al, 2008, GEO accession GSE10846), we found that miR-155 exhibited a reciprocal expression pattern with DEPTOR and c-CBL (r =-0.15; p=.002 and r=-0.189; p<.0001), indicating that miR-155 likely modulates expression of c-CBL and DEPTOR in primary tumors.

Since the role of DEPTOR in DLBCL has not been previously addressed, we investigated its expression in a series of 76 newly diagnosed DLBCL patients by immunohistochemistry. Complete loss of DEPTOR expression was noted solely in non-GCB tumors (9 of 37; 24%). Additional 12 patients exhibited low expression of this protein. In contrast, none of the 39 GCB tumors stained negative for DEPTOR (Chi-square, p=.005). DEPTOR mRNA greater than mean in primary DLBCL biopsies was associated with longer overall survival (OS; P=0.016).

To elucidate the function of DEPTOR in DLBCL cells, we silenced the expression of this protein with shRNA. Attenuated protein level of DEPTOR markedly enhanced AKT activity and promoted proliferation of DHL4 cells. DHL4 cells with silenced DEPTOR were also less prone to starvation- or SYK inhibition- induced apoptosis. Since AKT has been reported to stimulate activity of NFκB, we hypothesized that miR-155-induced decrease in DEPTOR expression would affect NFκB signaling. Consistent with this, inhibition of endogenous miR-155 in U2932 cells led to marked downregulation of NFĸB-controlled genes (CD40, BFL-1, RelB, IĸBα, A20, MIR155HG) and sensitized U2932 cells to ibrutinib, indicating that miR-155 amplifies NFĸB signaling in these cells.

Conclusions

Taken together, our data underscore the role of miR-155 in the regulation of AKT and NFkB prosurvival signaling in DLBCL. By targeting multiple negative regulators of PI3K/AKT pathway, miR-155 creates a feed - forward loop leading to increased NFkB activity. We also show that DEPTOR protein expression is decreased or lost selectively in a large fraction of ABC-DLBCLs. Since DEPTOR modulates AKT activity and its silencing promotes proliferation of DLBCL cells, these data suggest that DEPTOR functions as a tumor suppressor in ABC-DLBCLs.