NF-κB Independent Protection Against Apoptosis by Overexpression of A20 in B-Cells

In Activated B-Cell like Diffuse Large B-Cell Lymphomas (ABC DLBCLs) the most commonly affected gene is TNFAIP3, which encodes A20, an ubiquitin modifying enzyme involved in the negative regulation of NF-κB signaling. A20 is itself a target gene of NF-κB and needs to be induced in order to exert its negative feedback effect on NF-κB. Mutations resulting in the inactivation of A20 have been found in a significant proportion of ABC DLBCL cases (Compagno M et al, Nature 2009). The tumor suppressor role of A20 was established by the reintroduction of wild-type A20 in some mutant A20 lymphoma B-cell lines promoting apoptosis, supposedly due to the loss of the ability to inactivate NF-κB. However, a protective effect of A20 against apoptotic stimuli was demonstrated in some cell types. In endothelial cells, A20 protects from TNF- and Fas-mediated apoptosis by inhibiting caspase-8 activation (Daniel S et al, Blood 2004). We thus wanted to understand the relationship between A20 expression, NF-κB activation and protection against apoptosis in B-cells.

We have cloned cDNAs coding for wild-type A20 (A20wt), for IκBα_S32,36A (constitutively active form of IκBα), and for luciferase (as a control) into a doxycycline double-inducible episomal vector pRT-1, and stably transfected these vectors into an EBV positive lymphoblastoid B-cells line. Induction of p53 mediated apoptosis was performed by treatment of cells with Fludarabine*, whereas induction of caspase-8 dependant was performed with agonist antibodies against Fas (CD95).

Both A20 and IκBα_S32,36A overexpression suppressed NF-κB activity with downregulation of NF-κB target genes. Loss of NF-κB activation by induction of IκBα_S32,36A led to increase of spontaneous apoptosis. Oppositely, loss of NF-κB activation by induction of A20 led to decrease of spontaneous apoptosis. Compared with luciferase transfected cells, no effect of A20 overexpression was seen after treatment of cells with anti-CD95 antibodies. Caspase-9 cleavage was significantly decreased and increase of TP53 protein levels was lower in A20 overexpressing cells after Fludarabine* treatment. By contrast, Fludarabine* treatment of IκBα_S32,36A expressing cells led to an increase of both TP53 protein levels and caspase-9 cleavage. Therefore, A20 negatively modulated mitochondrial apoptosis pathway despite NF-κB downregulation.

These results suggest that A20 may exert an anti-apoptotic effect on its own independently of NF-κB activation. We are now looking after the functional properties of A20 natural mutants. Indeed, we can hypothesize that A20 mutants described in ABC DLBCLs have lost their negative feedback effect on NF-κB, but not their protective effect against apoptosis. So, A20 mutants may actively contribute to oncogenesis through their own protective effect on tumor cells.

No relevant conflicts of interest to declare.