A novel mechanism of CD40-induced apoptosis of carcinoma cells involving TRAF3 and JNK/AP-1 activation

Abstract

Membrane-presented CD40 agonists can induce apoptosis in carcinoma, but not normal homologous epithelial cells, whereas soluble agonists are growth inhibitory but not proapoptotic unless protein synthesis is blocked. Here we demonstrate that membrane-presented CD40 ligand (CD154) (mCD40L), but not soluble agonists, triggers cell death in malignant human urothelial cells via a direct mechanism involving rapid upregulation of TNFR-associated factor (TRAF)3 protein, without concomitant upregulation of TRAF3 mRNA, followed by activation of the c-Jun N-terminal kinase (JNK)/activator protein-1 (AP-1) pathway and induction of the caspase-9/caspase-3-associated intrinsic apoptotic machinery. TRAF3 knockdown abrogated JNK/AP-1 activation and prevented CD40-mediated apoptosis, whereas restoration of CD40 expression in CD40-negative carcinoma cells restored apoptotic susceptibility via the TRAF3/AP-1-dependent mechanism. In normal human urothelial cells, mCD40L did not trigger apoptosis, but induced rapid downregulation of TRAF2 and 3, thereby paralleling the situation in Blymphocytes. Thus, TRAF3 stabilization, JNK activation and caspase-9 induction define a novel pathway of CD40-mediated apoptosis in carcinoma cells.

CD40 Ligand-Induced Carcinoma Cell Death: A Balance between Activation of TNFR-Associated Factor (TRAF) 3-Dependent Death Signals and Suppression of TRAF6-Dependent Survival Signals

“…In agreement with our results, it has been reported that membranebound CD40L but not rsCD40L upregulated TRAF3 in urothelial cancer cells but not normal urothelium (9). However, we are the first to show that there is a reciprocal downregulation of TRAF6 in response to membrane bound CD40L.…”

“…Low levels of CD40 ligation promote cell survival/proliferation, whereas high levels induce growth arrest/ apoptosis (5-7). The precise form of the CD40 stimulus also affects these responses, with the most profound effects in carcinoma cells being induced by membrane-bound rather than recombinant soluble CD40L (rsCD40L) (8,9). The signaling pathways underlying these differential responses are yet to be fully characterized, and there is controversy in the field as to the relative contribution of CD40-induced activation of the JNK/AP-1 MAPK pathway versus upregulation of membrane-bound ligands of the TNF family in CD40L-mediated carcinoma cell apoptosis (9,10).…”

“…We have previously found that rsCD40L can stimulate survival-signaling pathways (including PI3K and ERK/MAPK) and induces apoptosis in carcinoma cell lines only in the presence of either the protein synthesis inhibitor cycloheximide, cytotoxic drugs, or inhibitors of the PI3K/mammalian target of rapamycin (mTOR) and/ or ERK pathways (16)(17)(18)(19). This contrasts with the effects of membrane-bound CD40L delivered by coculture of EJ cells with CD40L-expressing fibroblasts where robust apoptosis is observed in the absence of other agents (8,9). However, the mechanisms underpinning these differential responses remain to be fully elucidated.…”

Adenovirus delivery of human CD40 ligand gene confers direct therapeutic effects on carcinomas

“…To examine whether RAd-hCD40L infection results in CD40 stimulation, lysates from EJ cells infected with CD40L or b-galactosidase-expressing virus were analyzed for c-Jun N-terminal kinase (JNK) activation, a pathway known to be activated by soluble CD40L or agonistic CD40 mAb treatment of malignant cells. 10,30 As shown in Figure 1d, RAd-hCD40L induced sustained phosphorylation of JNK compared with rsCD40L protein that only transiently activated this pathway.…”

“…Concomitant with this idea, RAd-hCD40L but not soluble CD40L displays prolonged engagement of the JNK signaling pathway (Figure 1d), which has been implicated in the pro-apoptotic effects of CD40 ligation. 30 The death-inducing and inflammatory activity of membrane-tethered FasL, a well-studied TNF family member, is regulated by soluble FasL, which is shed from the membrane-bound form of the molecule by putative metalloproteinase(s). 32,33 RAd-FasL-infected cells secrete soluble FasL that reduces the cytotoxic effects and therapeutic capacity of this virus.…”