Microrna-155 Controls The RB/E2F Axis In Normal and Malignant B Cells Via The Non-Canonical TGFβ1-SMAD5 Signaling Module

MicroRNA-155 (miR-155) plays pleiotropic roles in the biology of normal and malignant B cells. MiR-155 knockout (KO) mice have fewer germinal center B cells, while overexpression of this miRNA is associated with aggressive DLBCL. Although several miR-155 targets have been identified, a mechanism that unifies the features of loss and gain of miR-155 function in normal and malignant cells remains to be described. In B cells, TGFβ signals are suppressive indicating that deregulation of this pathway may interfere with the developmental regulation of lymphocytes and contribute to the pathogenesis of B cell malignancies. Earlier, we described the direct targeting of the transcription factor SMAD5 by miR-155, and uncovered the presence of non-canonical signaling model in B cell lymphomas whereby TGFβ1, a cytokine that typically activates SMAD2/3, phosphorylated SMAD5. Herein, we used the miR-155 KO mice and genetically modified DLBCL cell lines to investigate which downstream effectors of TGFβ signals are disrupted by the miR-155/SMAD5 interaction, thus shedding light on the phenotypes associates with miR-155 loss and gain of function.

We confirmed the phosphorylation of SMAD5 by TGFβ1 in DLBCL cell lines, and demonstrated for the first time that this non-canonical signal is also present in untransformed normal mature B cells. We stably expressed miR-155 in the TGFβ1-responsive DLBCL cell lines Ly7, Ly18 and DHL5, and readily detected suppression of SMAD5, but not of other SMADs. TGFβ1 cytostatic activities include up-regulation of p15 and p21, which are primarily found in the context of SMAD2/3 activation. However, we found that stable expression of miR-155, and downregulation of SMAD5, significantly limited TGFβ1-dependent induction of both p15 and p21 in DLBCL. TGFβ1-mediated upregulation of p15 and p21 limits the activity of cyclin/CDK complexes, enriches for hypophosphorylated (active) RB, and promotes cell cycle arrest. We measured the effects of miR-155 in this process, and found that the accumulation of hypophosphorylated RB following TGFβ1 exposure was blunted in miR-155 expressing cells, resulting in an impaired G0/G1 arrest. The impact of miR-155 on TGFβ1 activity was also detectable by directly measuring the phosphorylation levels of RB’s Ser780 residue. Active pRB blocks cell cycle progression at least in part by binding to and inhibiting the E2F family of transcriptional regulators. Thus, we performed co-immunoprecipitation experiments and quantified the levels of RB-bound E2F1. In these assays, following TGFβ1 exposure we found a markedly decreased pRB-E2F1 complex formation in miR-155 expressing cells when compared to their controls. In agreement with these data, DLBCL cell lines expressing miR-155 displayed higher levels of free E2F1. Together, these data suggested the existence of a miR-155-SMAD5-p15/p21 axis that regulates TGFβ1 effects towards RB and E2F in DLBCL. To confirm the specific role of each component in this circuit, we used an RNAi strategy to transiently or stably knockdown (KD) SMAD5, p15 or p21 in our DLBCL models. In control RNAi cells, exposure to TGFβ1 led to decrease in RB phosphorylation, whereas these effects were abrogated upon KD of each of these genes, resulting in accumulation of hyperphosphorylated RB, a phenocopy of miR-155 expression. To define if the interplay between miR-155/SMAD5 and RB was also present in non-malignant cells, we purified mature B lymphocytes from miR-155 WT and KO mice. Examination of four pairs of mice, showed a higher expression of SMAD5 in cells from miR-155 KO than WT mice. In addition, TGFβ1-mediated suppression of phospho-RB was consistently more pronounced in miR-155 KO than in WT B cells, which resulted in a significantly higher G0/G1 arrest in cells lacking this miRNA. Of note, in absence of TGFβ1 there was no significant difference in cell cycle profile of mature B cells from miR-155 WT and KO mice. We concluded that an unrestrained TGFβ activity, secondary to SMAD5 upregulation, may help explain the deficient germinal center B cells formation found in miR-155 KO mice.

Together, our findings demonstrate that miR-155 overexpression is a novel model for deregulation of the lymphomagenic RB/E2F axis, and define an unsuspected role for the non-canonical TGFβ1 activation of SMAD5 in the developmental regulation of mature B cells.