Smoothened (SMO) Activates NF-Kb Pathway Through Activation of PKCβ/CARMA1 and TRAF6 Stabilization in Diffuse Large B-Cell Lymphoma

Abstract 1298 Aberrant activation of hedgehog (Hh) and NF-kB pathways contribute to tumor cell growth, survival and chemotolerance in diffuse large B-cell lymphoma (DLBCL). Previously, we documented a functional crosstalk between hedgehog (Hh) and NF-kB pathways that contribute to tumor cell growth and survival in diffuse large B-cell lymphoma (DLBCL). However, the molecular mechanisms that link Hh with NF-kB pathway have not been defined. Based on that smoothened (SMO) has been associated with heterotrimeric G protein members of the Gα i family and established as a GPCRs-like protein, we hypothesize that GPCRs-related mechanisms such as the protein kinase C (PKC)-CARMA1-BCL10/MALT1/TRAF6 axis may contribute to SMO-dependent activation of NF-kB. First, we confirmed that SMO contributes to the activity of total PKC in 293T and DLBCL cells. Transiently or stably silencing of SMO resulted in decreased total PKC activity in comparison with the controls, while overexpression of SMO resulted in increased activity of PKC. Cyclopamine-KAAD or recombinant Shh N-terminal peptide resulted in decrease or increase of the total activity of PKC, respectively. As PKC isoforms β-1 and β-2 are the major isoforms expressed in B-lymphocytes that mediates NF-kB activation induced by activation of the B-cell receptor, we assessed the activation status of these two isoforms in response to changes of SMO activity. Inhibiting SMO with cyclopamine-KAAD or silencing SMO by siRNA decreased the phosphorylation status of PKCβ-1 and −2. In contrast, activating SMO with Shh N-terminal peptide increased the phosphorylation of PKCβ-1 and −2. Next, we assessed if SMO can modulated the activity of CARMA1. CARMA1 is a scaffold protein that serves to integrate the upstream signal of PKCs with downstream effectors in hematopoietic cells. We found that cyclopamine-KAAD or silencing SMO by siRNA decreased the phosphorylation status (inactivation) of CARMA1 and that activation of SMO with Shh N-terminal peptide increased the phosphorylation (activation) of CARMA 1. Because the polyubiquitination of TRAF6 and NEMO (IKKg) at lysine 63 (K63) are important events in propagating NF-kB signaling we examined the effect of overexpressing SMO on K63 polyubiquitination of TRAF6 and NEMO. Overexpression of SMO resulted in increased polyubiquitination of TRAF6 and NEMO at K63 supporting NF-kB pathway activation. TRAF6 is involved in activation of TAK1 and the IKK complex, resulting in translocation of NF-kB to the nucleus and activation of NF-kB. Silencing SMO by shRNA decreased total levels of TRAF6 that was associated with faster proteosomal degradation of TRAF6 in 293T cells. Inhibition of SMO with cyclopamine-KAAD also decreased the total levels of TRAF6 in DLBCL cells. In addition, overexpression of SMO (including a constitutively active mutated SMO) in 293T cells caused increase of TRAF6 expression by decreased polyubiquitination of TRAF6 at lysine 48 (K48) that targets TRAF6 for proteosomal degradation. In summary, altogether, these findings support that at least one mechanism by which SMO contributes to modulate activation of NF-kB is through the activation of the axis PKCβ/CARMA1/TRAF6/NEMO and stabilization and blocking degradation of TRAF6. Disclosures: No relevant conflicts of interest to declare.