The immunological effects of multikinase inhibitors routinely used in cancer treatment are emerging. The present study was undertaken to investigate the potential ability of Sorafenib to manipulate cytokine expression by macrophages.
Many previous studies have shown the biological effects of PGE2
and other cAMP elevating substances on the cytokine profile of macrophages, namely the suppression of several inflammatory cytokines and enhancement of the anti-inflammatory cytokine IL-10[14
]. We wished to investigate the impact that Sorafenib could potentially have in the presence of PGE2
and other cAMP elevating extracellular mediators. We determined that Sorafenib could restore the expression of IL-12 and suppress IL-10 in a dose-dependent manner. Furthermore, activation with LPS alone in the presence of Sorafenib led to high levels of IL-12 secretion. These data suggest that Sorafenib interferes with a global mechanism by which inflammatory cytokine expression is suppressed and IL-10 expression is induced in macrophages.
The mechanism of PGE2
-induced suppression of inflammatory cytokines has partially been elucidated. PGE2
has been shown to partially inhibit LPS-induced degradation of NF-κB p105 via the prostaglandin E receptor EP4. This mechanism is responsible for the inhibition of TNF-α, MCP-1, and a number of other cytokines[31
]. It does not appear to be responsible for the inhibition of IL-12p40[31
]. We have shown that Sorafenib reverses the inhibition of IL-12p40 mediated by the presence of PGE2
, but not the inhibition of TNF-α (data not shown). Furthermore, we have observed that the degradation of p105 that is inhibited by PGE2
is not restored by the presence of Sorafenib (data not shown).
Two pathways that can differentially regulate inflammatory versus anti-inflammatory cytokine production are the MSK and GSK3-β kinase pathways. Interrogating the p38 and ERK MAP kinase signaling pathways revealed that Sorafenib can inhibit p38 activation and activation of its downstream target protein kinase MSK while having no effect on the activation of ERK. The lack of an effect on the activation of ERK, however, is not entirely unexpected as LPS induced activation of ERK1/2 is through a RAF-independent mechanism via the MAP3K TPL-2 (MAP3K8 [32
]). Furthermore, inhibition of p38/MSK pathway disrupted the phosphorylation of histone H3 at serine 10, a downstream phosphorylation target of the MSKs. The MAPK p38α is integral in dampening inflammation via the MSKs in macrophages and other cells of myeloid origin [33
]. Furthermore, the deletion of p38α leads to enhanced expression of a number of pro-inflammatory mediators, including IL-12p40, and severely diminished expression of anti-inflammatory IL-10[33
]. The MSKs have been previously described as negative regulators of Toll-like receptor signaling and integral to regulating excessive expression of inflammatory cytokines, including IL-12p40. In addition, they have been shown to be required for transcription factor association to the il10
]. Inhibition of p38/MSK appears to a be a major mechanism contributing to the ability of Sorafenib to promote excessive IL-12p40 expression in LPS simulated macrophages and restoring its expression in macrophages stimulated with LPS+ PGE2
via an IL-10 independent mechanism. Furthermore, inhibition of the MSKs also provides a potential mechanism for the inhibition of IL-10 expression.
Cytokine expression induced by Toll-like receptor engagement has previously been shown to be differentially regulated by glycogen synthase kinase (GSK) 3-β. GSK3-β is a constitutively active downstream kinase of the PI3K/Akt pathway which is inactivated upon phosphorylation at Ser9[27
]. Direct inhibition of GSK3-β via the presence of the inhibitors LiCl or SB216763 diminishes the expression of IL-12p40 and enhances IL-10 production. Interference with AKT meditated inhibition of GSK3-β activity via Akt or PI3K inhibitors led to enhanced expression of IL-12p40 and suppression of IL-10 expression[27
]. As we saw a similar pattern with macrophages stimulated in the presence of Sorafenib, we investigated the potential inhibitory activity of Sorafenib on the inactivation of GSK3-β. Sorafenib did show modest inhibition of both AKT activation and GSK3-β phosphorylation when macrophages were stimulated with LPS+PGE2
. However, inhibition of AKT prior to stimulation with LPS+PGE2
did not lead to the restoration of IL-12p40 expression. Therefore, inhibition of the Akt/GSK3-β did not appear to the major mechanism leading to the restoration of IL-12p40 expression.
Due to the promiscuity of Sorafenib as an inhibitor it may have some unintentional targets which could boost its potential for successful anti-cancer therapy. Tumor associated macrophages have increasingly been recognized as tumor promoting. They appear to share many properties with regulatory macrophages and aid in tumor metastasis, tumor growth, down-regulation of adaptive immunity, and further drive the differentiation of recruited monocytes to a regulatory-like phenotype. They produce abundant IL-10 and are devoid of IL-12[34
]. For certain tumors it is possible that Sorafenib may not only contribute to tumor resolution though its established mechanisms of vascular endothelial growth factor receptor (VEGFR) signaling blockade and direct tumor toxicity, but potentially also by transitioning macrophages from an regulatory-like to tumor-resolving inflammatory phenotype via the suppression of IL-10 and restoration of IL-12 production. The restoration of IL-12 and inhibition of IL-10 expression by tumor associated macrophages have been considered to be potentially valuable anticancer targets which could potentially have a profound impact on the tumor microenvironment[35
]. Enhancing the presence of IL-12 within the tumor environment has been shown to contribute to tumor clearance through a variety of mechanisms, including restoring the cytotoxicity of tumor-resident CD8+ T-cells[36
] and stimulating IFN-γ mediated inhibition of tumor-induced regulatory T-cell proliferation[38
]. Future studies addressing the use of Sorafenib either in mono- or combinatorial therapy should potentially examine the influence it may have on macrophages within the tumor environment in addition to its well established tumoricidal and anti-angiogenic effects.