For years, a primary goal of tumor immunologists has been to trigger an anticancer response by the patient's own immune system, directed largely at engaging the adaptive immune system to mount a tumor-specific response (1
). However, a considerable body of evidence suggests that nonlymphocytic immune cells also play an important role in eradicating tumors (4
). A new class of low molecular mass chemotherapeutic agents, vascular disrupting agents (VDAs), stimulate a variety of cell types, including cells of the monocyte/macrophage lineage, to undergo morphological and functional changes that lead to cytokine release, increased vascular permeability, and rapid and sustained tumor vascular collapse (6
One class of VDAs includes flavone acetic acid and its derivatives, e.g., 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Although flavone acetic acid was found to exert extraordinary antitumor effects in mice, failed clinical trials revealed the species-specific nature of this compound (10
). In contrast, DMXAA is currently in advanced phase II clinical trials and has shown great promise in the treatment of a variety of malignancies (12
). The molecular mechanisms of action of flavonoid VDAs are largely unknown; however, induction of cytokines has been implicated as a proximal event by which these agents induce tumor necrosis (14
Early studies revealed differences in gene induction patterns elicited in mouse macrophages stimulated by DMXAA versus the highly potent Toll-like receptor 4 (TLR4) agonist, Escherichia coli
). Perera et al. reported that DMXAA potently induced a subset of LPS-inducible genes that included both IFN-inducible protein 10 (IP-10) and IFN-β but poorly induced expression of proinflammatory genes such as TNF-α (7
). Although TNF-α was initially suspected to induce tumor necrosis after DMXAA, TNF-α receptor–deficient mice displayed only a partially diminished capacity to reject tumor explants when treated with DMXAA, and serum from human subjects treated with DMXAA contained no detectable TNF-α (17
). Jassar et al. later showed that macrophages are among the first cells to infiltrate the tumor after DMXAA treatment and are responsible for secreting large amounts of cytokines (19
). Moreover, they express high levels of chemokines that may recruit cells into the tumor. Although the mechanism of action of DMXAA remains unknown, it is apparent from these studies that the macrophage response to DMXAA is important and requires further clarification.
Major advances have led to a detailed understanding of many of the signaling molecules involved in activation of the cells of the innate immune system (20
). Among these, TLRs compose a major receptor family that enables pathogens to be sensed by the host. TLRs are expressed either on the surface or on an endosomal membrane of immune cells, where they detect conserved pathogen-associated molecular patterns (PAMPs). PAMP-induced oligomerization of TLRs recruits intracellular adaptor molecules to the C-terminal domain. Differential engagement of PAMPs through the N terminus, coupled with differential recruitment and utilization of individual adaptor molecules by the different TLRs, provides the basis for the specificity with which cells respond to different PAMPs with different patterns of gene expression (21
To date, four adaptors (myeloid differentiation factor 88 [MyD88], Toll–IL-1 resistance [TIR] domain–containing adaptor protein, TRIF-related adaptor molecule [TRAM], and TIR domain–containing adaptor inducing IFN-β [TRIF] [22
]) have been associated with TLR signaling. MyD88 is absolutely required for the response to PAMPs detected by all known TLRs, with the exception of TLRs 3 and 4 (22
). In the case of TLR4, all four adaptors are used, and the intracellular signaling cascade bifurcates into MyD88-dependent (i.e., MyD88 and TIR domain–containing adaptor protein mediated) and MyD88-independent (i.e., TRAM and TRIF mediated) arms (26
). MyD88-dependent signaling leads to rapid recruitment of the family of IL-1R–associated kinases, phosphorylation of inhibitor of κB (IκB) α, nuclear translocation of NF-κB, and expression of proinflammatory genes such as TNF-α and IL-1β (20
). In the case of TLR4, the MyD88-independent pathway utilizes TRAM to recruit TRIF that, in turn, recruits two noncannonical IκB kinases (IKKs), TANK-binding kinase 1 (TBK1) and IKK
). Both phosphorylate the transcription factor IFN regulatory factor 3 (IRF-3) and result in a later wave of NF-κB translocation (31
). Once phosphorylated, IRF-3 and NF-κB translocate to the nucleus, where they activate genes such as IFN-β.
In 2004, Yoneyama et al. described a TLR-independent pathway leading to IFN-β expression (32
). Rather than a TLR, a cytosolic RNA helicase, retinoic acid–inducible gene I (RIG-I), detects double-stranded viral RNA via its helicase domain. RIG-I binds to an adaptor molecule, IFN-β promoter-stimulator 1 (IPS-1; also known as MAVS, Cardif, and VISA), that leads to TBK1/IKK
activation, IRF-3 phosphorylation, and transcription of IFN-β (33
). Another RIG-I–like molecule, melanoma differentiation–associated gene 5, has also been previously described (34
). RIG-I and melanoma differentiation–associated gene 5 distinguish between different RNA viruses, but both use IPS-1 (35
). Stetson et al. recently described yet another pathway leading to IRF-3 activation (37
). Although the molecular sensor was not identified, cytosolic DNA was found to activate IRF-3 and induce IFN-β in the absence of detectable NF-κB or mitogen-activated protein kinase (MAPK) activation.
In this study, we detail a novel IFN-β–inducing pathway that is activated by DMXAA. DMXAA dramatically up-regulates IRF-3–dependent gene expression in a TLR- and IPS-1–independent manner. The response was completely dependent on both TBK1 and IRF-3 but elicited no detectable MAPK activation and minimal, delayed NF-κB DNA binding activity. Additionally, we show that although DMXAA does not lead to measurable IκBα degradation, it results in phosphorylation of p65 in a TBK1-dependent, but IKKβ-independent, manner. We also find that pretreatment of macrophages with either DMXAA or LPS induces a state of “cross-tolerance” for subsequent stimulation by DMXAA or LPS, suggesting shared utilization of signaling molecules. Interestingly, we also show that salicylic acid (SA) inhibits DMXAA- but not LPS-induced IRF-3 signaling in macrophages. Collectively, these data establish DMXAA as a novel, potent, and specific activator of the TBK1–IRF-3 signaling cascade.