Targeting of SMAD5 Links MicroRNA-155 to the TGFβ Pathway and Lymphomagenesis.

Abstract 315

The mechanisms by which microRNA (miRNA) dysfunction contribute to the pathogenesis of diffuse large B-cell lymphoma (DLBCL) are not well established. MiRNAs are small non-protein coding RNAs that function by regulating the expression of target transcripts. Thus, to capture the physiologic and pathologic impact of these small regulatory molecules it is necessary to identify the genes that they inhibit. MicroRNA-155 (miR-155) is overexpressed in aggressive subsets of DLBCL, and its enforced expression in a transgenic, Eμ-miR-155, mouse model is associated with the development of lymphoblastic leukemia/high grade lymphoma. However, although a series of bona-fide miR-155 targets have been identified, it is still unclear how overexpression of this miR-155 contributes to lymphomagenesis. Starting from unbiased genome-wide approaches in DLBCL, we discovered that miR-155 directly binds to two sites in the 3'UTR of the SMAD5 gene, inhibiting its expression. Surprisingly, although SMAD5 activity is classically associated with signals transduced by bone morphogenic protein (BMP) family of cytokines, we found that in DLBCLs a non-canonical signaling module linking TGFβ1 to SMAD5 is also active. Furthermore, using kinase inhibitors specific to BMP or TGFβ receptors, we found that the type I TGFβ receptors, in particular ALK5, were essential for the activation of SMAD5 by TGFβ1 in DLBCL. These data suggested that the impact of miR-155 on the TGFβ pathway could be broader than previously appreciated, and abrogate the growth inhibitory effects of both BMP and TGFβ1 in DLBCL. To test this hypothesis, we generated DLBCL cell lines (Ly1, Ly18 and Ly19) ectopically expressing miR-155 and found that they became resistant to the cytostatic effects of TGFβ1 and BMPs. This blockade in growth inhibition was associated with an impaired cell cycle arrest and defective induction of p21. Next, to firmly establish the role of SMAD5 targeting in the miR-155-mediated disruption of the TGFβ signals, we created DLBCL cell lines stably expressing two independent SMAD5-shRNA constructs. Indeed, knockdown of SMAD5 in DLBCL limited the growth inhibitory effects of BMP and TGFβ1 and abrogated p21 induction, largely recapitulating the effects of miR-155 expression. In addition, using a xenograft model of DLBCL with in vivo luminescent imaging capabilities, we found that miR-155 overexpression yielded larger and more widespread lymphomas. Confirming our in vitro data, DLBCLs with stable SMAD5 knockdown were also more aggressive, indicating that SMAD5 downregulation phenocopies the effects of miR-155 in vivo. To gain further insights into the functional consequences of the miR-155/SMAD5 interplay, we used real-time RT-PCR to measure the transcriptional activation of several SMAD5 direct targets. In DLBCL cell lines ectopically expressing miR-155 or SMAD5 shRNA constructs, we found that the expression of the SMAD5 targets, ID1, ID2, SMAD6, SMAD7, BMPR2 and BMP6, was significantly diminished. These data further stressed the relevance of miR-155-mediated SMAD5 downregulation, and started to unveil the downstream components of SMAD5's tumor suppressive activities. Finally, we expanded our observations to primary tumors, and used western blotting to show that miR-155 overexpressing DLBCLs had significantly lower levels of SMAD5, and exhibited an impaired expression of its transcriptional targets. Together, our data instructed on miR-155 mediated lymphomagenesis, highlighted a hitherto unappreciated role of SMAD5 as a lymphoma suppressor gene, and defined a novel mechanism used by cancer cells to escape TGFβ growth inhibitory effects.