TNFR family members convey signals leading to the regulation of diverse cellular responses, ranging from proliferation and differentiation to growth suppression and apoptosis (12
). Among these receptors, TNFR1, Fas, TRAIL-R1, TRAIL-R2, and DR3 share death domain homology in their cytoplasmic tails, through which they transduce apoptotic signals. Paradoxically, other members of the TNFR superfamily which lack the death domain in their cytoplasmic regions, such as TNFR2, CD30, and CD40, have also been reported to suppress growth and survival in a number of carcinoma cell lines (15
In this study, we have investigated the mechanism by which CD40 induces cell death in carcinomas. For this purpose, we have exposed human carcinoma cell lines to recombinant soluble forms of CD40L and found that induction of cell death depends on the oligomerization status of CD40L. Thus, treatment with CD40L monomers had no effect on survival, but apoptosis was induced following antibody-induced monomer cross-linking, which leads to ligand dimerization. Cell death was even more pronounced following treatment with trimeric rsCD40L (Fig. A and B), a phenomenon which probably reflects differences in the efficacy of these molecules to aggregate CD40 (20
). Consistent with our findings, previous studies, including the recent identification of the crystal structure of the CD40-TRAF2 complex, have emphasized the significance of ligand-mediated trimerization for efficient CD40 signalling (7
). While oligomerization of CD40 is necessary for transduction of signals which activate the cell death machinery in carcinoma cells, its execution also requires inhibition of protein synthesis by CHX, in common with the effects of anti-Fas or TNF treatment in carcinoma cell lines (9
). It is possible that CHX blocks the production of protective antiapoptotic proteins, thereby unmasking the cytotoxic potential of CD40 activation. Indeed, the interactions of CD40 with its ligand have been shown to induce the transcriptional up-regulation of a number of negative regulators of cell death, such as Bcl-xL, Bfl1, and A20 (10
The requirement of protein synthesis inhibition for efficient killing also unveils a mechanism by which tumor cells, through the activation of antiapoptotic programs such as the reported constitutive activation of phosphatidyl inositol 3-kinase–Akt and Bcl-2 overexpression in a subset of ovarian tumors (8
), may escape CD40-mediated cytotoxicity. While the ability of these pathways to block CD40-mediated cell death remains to be verified, previous work has implicated Bcl-2, Bcl-xL, and Akt in suppression of TNF- and Fas-induced apoptosis in certain cell types (5
). Furthermore, CD40 is absent in a proportion of tumors of the breast and cervix as well as in a number of tumor cell lines, such as the cervical HeLa and ME180, the MCF7 breast, and the 2780CP ovarian carcinoma cell lines, suggesting possible selection for CD40-negative cells (15
; Eliopoulos and Young, unpublished observations). An alternative mechanism of resistance to CD40-mediated cell death may occur through disruption of CD40L-induced signals. This is exemplified by the inability of A2780 ovarian carcinoma cells to respond to CD40-mediated cell death (Fig. A). In these cells, NF-κB but not JNK activation in response to CD40 stimulation appears to be impaired (N. J. Gallagher, A. G. Eliopoulos, et al., unpublished data). This is not peculiar to A2780 cells, as CD40 ligation fails to induce NF-κB-dependent transcription in Hodgkin's cell lines (71
) and similar deficiencies have been identified in a mouse pre-B-cell line (13
). While NF-κB has been implicated in the generation of protective responses against TNF- and drug-induced cell death (68
), its contribution as a proapoptotic signal has also been noted (3
). Indeed, anticancer drugs are known to induce FasL, as well as Fas expression, through a mechanism which critically involves activation of NF-κB (38
). In addition, inhibition of NF-κB by a constitutively active IκBα has been recently shown to suppress phorbol myristate acetate- and ionomycin-induced FasL expression and apoptosis in Jurkat T cells (45
), and NF-κB is a positive regulator of serum withdrawal-induced apoptosis in 293 cells (28
). Interestingly, while A2780 cells do not undergo apoptosis in response to CD40 ligation, we have previously shown that their long-term exposure to CD40L in the absence of CHX leads to growth inhibition (15
). This phenomenon is reminiscent of the antiproliferative properties of CD40 ligation in Burkitt's lymphoma cell lines, in the absence of an effect on viability (2
). Therefore, CD40 engagement in tumor cells may activate two distinct pathways, leading to inhibition of proliferation or induction of cell death. The signalling cascades which regulate these CD40 pathways will be an interesting area for future studies.
Mutational analysis of the CD40 cytoplasmic tail demonstrated that the TRAF2- and TRAF3-interacting PXQXT motif, a major CD40 signalling effector site, is not critical for induction of apoptosis but death signals are transduced through its membrane-proximal domain. This region binds TRAF6, which is a known regulator of NF-κB, JNK, and ERK signals by CD40 (35
). While a role for TRAF6 in modulating cell death has not been described, TRAF6 but not TRAF2 or TRAF3 interacts with the NF-κB and apoptosis-inducing protein RIP2 (48
). The contribution of TRAF6 and RIP2 to CD40-mediated cytotoxicity is currently under investigation. Intriguingly, CD40 signals generated from its membrane-proximal, TRAF6-interacting domain appear to be qualitatively different from those engaged by the PXQXT motif. Thus, ERK activation by the membrane-proximal region is Ras independent, whereas that by the PXQXT is Ras dependent (37
), and recent evidence suggests differential regulation of NF-κB by these two CD40 domains (66
). Furthermore, JAK or STAT signalling is engaged exclusively from the membrane-proximal region (29
), and our work provides further evidence for differential signalling emanating from these two domains.
The ability of CD40 ligation to confer a reduced and delayed apoptotic response compared to Fas stimulation, coupled with previous reports demonstrating a synergistic role for CD40 in anti-Fas-induced cytotoxicity (15
), prompted us to investigate the possibility that CD40-mediated apoptosis in carcinoma cells occurs indirectly via a mechanism involving Fas and/or its ligand. Three pieces of evidence corroborate this hypothesis. Firstly, we have shown that CD40L-induced cell death occurs through a crmA-sensitive, caspase-dependent pathway (Fig. and ), in keeping with the ability of Fas to engage a proapoptotic cascade that leads to caspase activation and is suppressed by crmA (63
). Furthermore, we have found that CD40 ligation induces the expression of Fas and FasL in apoptosis-susceptible cell lines (Fig. ). Finally, we have demonstrated that CD40L-induced apoptosis in carcinoma cell lines is partially inhibited by reagents which neutralize FasL (Fig. ).
The inability of neutralizing anti-FasL antibodies to completely abolish CD40-mediated cytotoxicity implies the contribution of additional death signals. Indeed, exposure of HeLa/CD40 cells to rsCD40L was also found to mediate transcriptional activation of other cytotoxic members of the TNF family, such as TRAIL (Apo-2L) and TNF, although with different kinetics. Consequently, we have found that a neutralizing anti-TNF antibody (27
) or a soluble TRAILR1:Fc partially protects against CD40-mediated cell death and that combination treatment with these neutralizing reagents has an additive effect on the survival of CD40L-treated cells. Therefore, CD40 ligation may induce apoptosis in susceptible carcinoma cell lines via an indirect pathway targeting more than one cytotoxic ligand of the TNF family. Whether CD40 ligation regulates the redistribution of intracellular cytoplasmic pools of these cytotoxic ligands, in addition to their de novo transcription and translation remains to be elucidated. Interestingly, our preliminary results indicate the presence of a preexisting cytoplasmic FasL pool in HeLa/CD40 cells which is significantly enriched following treatment with rsCD40L. This is consistent with previous studies in human carcinoma cells, monocytes, and T cells demonstrating the presence of high intracellular levels of FasL or TRAIL which rapidly translocate to the cell surface in response to various stimuli (41
). These observations may explain the ability of neutralizing FasL or TRAIL to suppress CD40L-induced apoptosis in carcinoma cells even in the absence of de novo protein synthesis.
The function of many TNF/TNFR family members appears to be tightly controlled in vivo partly through regulation of their expression. For example, Fas and CD40 are widely expressed, but their ligands are restricted to activated T cells and sites of immune privilege. Conversely, TWEAK is expressed in a number of tissues, but its receptor, DR3, is found only in lymphoid cells. The restricted expression of CD40L in vivo, coupled with its antiproliferative and preapoptotic properties when applied as a soluble form, makes it a suitable candidate for tumour therapy. This is supported by the ability of CD40 ligation alone to reduce growth and survival in early-passage ovarian carcinoma cells cultured in vitro (our unpublished observations) and by a recent study demonstrating significant breast tumor regression and apoptosis in xeno-transplanted SCID mice treated with rsCD40L (32
). While the mechanism of CD40-mediated carcinoma cell death in vivo is currently unknown, it is likely to involve activation of FasL and/or other death receptor ligands. In addition to regulating CD40-mediated cytotoxicity, these ligands and/or their receptors are also important in apoptosis induced by a broad spectrum of stimuli, including chemotherapy, radiation, ectopic c-myc expression, and anoikis (21
), further emphasizing their extensive and central role in programmed cell death.