Shiro Kanegasaki

On the basis of our recent findings, in which multiple receptor‐mediated mast cell functions are regulated via a common signaling cascade, we posit that the formation and functioning of osteoclasts are also controlled by a similar common mechanism. These cells are derived from the same granulocyte/monocyte progenitors and share multiple receptors except those that are cell‐specific. In both types of cells, all known receptors reside in lipid rafts, form multiprotein complexes with recruited signaling molecules, and are internalized upon receptor engagement. Signal transduction proceeds in a chain of protein phosphorylations, where adaptor protein LAT (linker‐for‐activation‐of‐T‐cells) plays a central role. The key kinase that associates LAT phosphorylation and lipid raft internalization is Syk (spleen‐tyrosine‐kinase) and/or an Src‐family‐kinase, most probably Lck (lymphocyte‐specific‐protein‐tyrosine‐kinase). Dephosphorylation of phosphorylated Syk and Lck by activated SHP‐1 (Src‐homology‐region‐2‐domain‐containing‐phosphatase‐1) terminates the signal transduction and endocytosis of receptors, resulting in inhibition of osteoclast differentiation and other functions. In malignant plasma cells (MM cells) too, SHP‐1 plays a similar indispensable role in controlling signal transduction required for survival and proliferation, though BLNK (B‐cell‐linker‐protein), a functional equivalent of LAT and SLP‐76 (SH2‐domain‐containing‐leukocyte‐protein‐of‐76‐kDa) in B cells, is used instead of LAT. In both osteoclasts and MM cells, therefore, activated SHP‐1 acts negatively in receptor‐mediated cellular functions. In osteoblasts, however, activated SHP‐1 promotes differentiation, osteocalcin generation, and mineralization by preventing both downregulation of transcription factors, such as Ostrix and Runx2, and degradation of β‐catenin required for activation of the transcription factors. SHP‐1 is activated by tyrosine phosphorylation and micromolar doses (M‐dose) of CCRI‐ligand‐induced SHP‐1 activation. Small molecular compounds, such as A770041, Sorafenib, Nitedanib, and Dovitinib, relieve the autoinhibitory conformation. Activation of SHP‐1 by M‐dose CCRI ligands or the compounds described may prevent the progression of bone lesions in MM.

Tumor-associated macrophages affect tumor progression and resistance to immune checkpoint therapy. Here, we identify the chemokine signal regulator FROUNT as a target to control tumor-associated macrophages. The low level FROUNT expression in patients with cancer correlates with better clinical outcomes. Frount-deficiency markedly reduces tumor progression and decreases macrophage tumor-promoting activity. FROUNT is highly expressed in macrophages, and its myeloid-specific deletion impairs tumor growth. Further, the anti-alcoholism drug disulfiram (DSF) acts as a potent inhibitor of FROUNT. DSF interferes with FROUNT-chemokine receptor interactions via direct binding to a specific site of the chemokine receptor-binding domain of FROUNT, leading to inhibition of macrophage responses. DSF monotherapy reduces tumor progression and decreases macrophage tumor-promoting activity, as seen in the case of Frount-deficiency. Moreover, co-treatment with DSF and an immune checkpoint antibody synergistically inhibits tumor growth. Thus, inhibition of FROUNT by DSF represents a promising strategy for macrophage-targeted cancer therapy.

Background: Signal transduction pathways mediated by various receptors expressed on mast cells are thought to be complex, and inhibitory signals that turn off activating signals are not known.Methods: Upstream signaling cascades mediated by several known receptors in bone marrow‐derived mast cells that lead to degranulation and mediator release were studied by immunoblotting and immunoprecipitation. Small interfering RNAs and knockout mice were used to confirm findings.Results: All ligands tested including IgE/Ag, SCF, HSP70, CCL3, and its valiant eMIP induced phosphorylation of linker for activation of T cells (LAT), which triggered their receptor‐mediated downstream signaling cascades that controlled degranulation and mediator release. Phosphorylation of lymphocyte‐specific protein kinase (Lck) was induced by each ligand, which commonly played an indispensable role in LAT phosphorylation. In contrast, phosphorylation of spleen tyrosine kinase was additionally induced in cells stimulated only with IgE/Ag and SCF, which is also associated with LAT phosphorylation in part. Degranulation and mediator release induced by IgE/Ag, SCF, or HSP70 were enhanced by nanomolar doses of CCR1 ligands CCL3 and eMIP via enhanced LAT phosphorylation. On the other hand, micromolar doses of CCR1 ligand inhibited degranulation and mediator release from mast cells stimulated with IgE/Ag, SCF, or HSP70 by de‐phosphorylation of phosphorylated Lck with Src homology region 2 domain‐containing phosphatase‐1.Conclusions: Linker for activation of T cells plays a central role in signal transduction pathways in mast cells stimulated with any ligand tested. Dose‐dependent alternate costimulation and inhibition of CCR1 ligands in IgE/Ag‐, SCF‐, or HSP70‐stimulated mast cells occur at the level of Lck‐LAT phosphorylation.

Significant numbers of malignant tumor cells that have spread to surrounding tissues and other distant organs are often too small to be picked up in a diagnostic test, and prevention of even such small metastases should improve patient outcomes. Using a mouse model, we show in this article that intravenous administration of a human CCL3 variant carrying a single amino acid substitution after mild local hyperthermia not only induces tumor growth inhibition at the treated site but also inhibits metastasis. Colon26 adenocarcinoma cells (1 × 10⁵ cells/mouse) were grafted subcutaneously into the right hind leg of syngeneic BALB/c mice and after nine days, when tumor size reached ~11 mm in diameter, the local tumor mass was exposed to high-frequency waves, by which intratumoral temperature was maintained at 42 °C for 30 min. Mice received the CCL3 variant named eMIP (2 μg/mouse/day) intravenously for five consecutive days starting one day after heat treatment. We found that tumor growth in eMIP recipients after hyperthermia was inhibited markedly but no effect was seen in animals treated with either hyperthermia or eMIP alone. Furthermore, the number of lung metastases evaluated at 18 days after hyperthermia treatment was dramatically reduced in animals receiving the combination therapy compared with all other controls. These results encourage future clinical application of this combination therapy.

To evade the immune system, the lethal human pathogen Streptococcus pyogenes produces SpyCEP, an enzyme that cleaves the C-terminal α-helix of CXCL8, resulting in markedly impaired recruitment of neutrophils to sites of invasive infection. The basis for chemokine inactivation by SpyCEP is, however, poorly understood, as the core domain of CXCL8 known to interact with CXCL8 receptors is unaffected by enzymatic cleavage. We examined the in vitro migration of human neutrophils and observed that their ability to efficiently navigate a CXCL8 gradient was compromised following CXCL8 cleavage by SpyCEP. SpyCEP-mediated cleavage of CXCL8 also impaired CXCL8-induced migration of transfectants expressing the human chemokine receptors CXCR1 or CXCR2. Despite possessing an intact N terminus and preserved disulfide bonds, SpyCEP-cleaved CXCL8 had impaired binding to both CXCR1 and CXCR2, pointing to a requirement for the C-terminal α-helix. SpyCEP-cleaved CXCL8 had similarly impaired binding to the glycosaminoglycan heparin. Enzymatic removal of neutrophil glycosaminoglycans was observed to ablate neutrophil navigation of a CXCL8 gradient, whereas navigation of an fMLF gradient remained largely intact. We conclude, therefore, that SpyCEP cleavage of CXCL8 results in chemokine inactivation because of a requirement for glycosaminoglycan binding in productive chemokine:receptor interactions. This may inform strategies to inhibit the activity of SpyCEP, but may also influence future approaches to inhibit unwanted chemokine-induced inflammation. Key Points SpyCEP-cleaved CXCL8 is unable to bind and activate CXCL8 receptors. Neutrophil glycosaminoglycans are required for migration along a CXCL8 gradient.

In preclinical studies with model animals, intravenous administration of a derivative of chemokine CCL3, named eMIP, after local electron-beam irradiation, not only enhanced tumor growth inhibition at a target site but also induced tumor killing beyond the treated site (a phenomenon known as the abscopal effect). eMIP works with alarmins such as high mobility group box 1 (HMGB1) and heat shock protein 70 (HSP70) released from overexpressed tumor cells by irradiation. These alarmins at the irradiated tumor bed trap injected eMIP and, by forming complexes with eMIP, play a key role to recruit and activate tumor inhibitory natural killer (NK) cells and CD4(+) and CD8(+) T cells. Tumor type-specific secretion of gamma interferon from splenocytes was also demonstrated, which may also activate NK cells. During Phase 1 clinical studies using X-rays, however, no apparent abscopal effect was observed. Instead, we saw frequent reduction in numbers of lymphocytes in the peripheral blood of irradiated patients. The reduced number of lymphocytes recovered poorly once depleted, in contrast to neutrophils, and persisted for months after the treatment. This might have affected outcome after combination treatment of irradiation and eMIP. To enhance host defense mechanisms during and after photon-beam (X-ray) radiotherapy of a deep-seated tumor, it seems essential to keep lymphocytes undamaged by eliminating reactive oxygen species that are formed in the peripheral blood during irradiation.

To evade the immune system, the lethal human pathogen Streptococcus pyogenes produces SpyCEP, an enzyme that cleaves the C-terminal α -helix of CXCL8, resulting in markedly impaired recruitment of neutrophils to sites of invasive infection. The basis for chemokine inactivation by SpyCEP is, however, poorly understood, as the core domain of CXCL8 known to interact with CXCL8 receptors is unaffected by enzymatic cleavage. We examined the in vitro migration of human neutrophils and observed that their ability to efficiently navigate a CXCL8 gradient was compromised following CXCL8 cleavage by SpyCEP. SpyCEP-mediated cleavage of CXCL8 also impaired CXCL8-induced migration of transfectants expressing the human chemokine receptors CXCR1 or CXCR2. Despite possessing an intact N-terminus and preserved disulphide bonds, SpyCEP-cleaved CXCL8 had impaired binding to both CXCR1 and CXCR2, pointing to a requirement for the C terminal α -helix. SpyCEP-cleaved CXCL8 had similarly impaired binding to the glycosaminoglycan heparin. Enzymatic removal of neutrophil glycosaminoglycans was observed to ablate neutrophil navigation of a CXCL8 gradient, whilst navigation of an fMLP gradient remained largely intact. We conclude therefore, that SpyCEP cleavage of CXCL8 results in chemokine inactivation due to a requirement for glycosaminoglycan binding in productive chemokine:receptor interactions. This may inform strategies to inhibit the activity of SpyCEP, but may also influence future approaches to inhibit unwanted chemokine-induced inflammation.

Background: In mast cells, induction of HSP70 expression during antigen stimulation has not been reported.Methods: Mouse bone marrow‐derived mast cells (BMMC) were stimulated with IgE/Ag or HSP70. Induction of HSP70 expression and signaling protein phosphorylation were evaluated by immunoblotting.Results: HSP70 expression is induced in BMMC at an early stage of IgE/Ag‐dependent stimulation, some of which is released from the cells in a granule‐associated form. Induction of HSP70 expression was also observed with an IgE/Ag‐stimulated human basophilic cell line, indicating that the phenomenon is not restricted to mouse BMMC. The induction of HSP70 expression, and its release, followed a similar time course to that of degranulation. Released HSP70 seems to be responsible for degranulation and production of eicosanoids, at least in part, because a neutralizing anti‐HSP70 antibody mitigated these activities and because exogenous HSP70 not only induced immediate degranulation followed by autocrine HSP70 expression but also enhanced degranulation in IgE/Ag‐stimulated BMMC. Extracellular HSP70 was found to induce phosphorylation of linker for activation of T cells (LAT) and a series of downstream signaling molecules in BMMC. We further found that Fyn, Lyn, and spleen tyrosine kinase (Syk), which are known to concern LAT phosphorylation in IgE/Ag‐stimulated BMMC, were not phosphorylated in HSP70‐stimulated BMMC, whereas lymphocyte‐specific protein tyrosine kinase (Lck) was phosphorylated.Conclusion: FcεRI stimulation in BMMC and basophils induces HSP70 expression and its release. Extracellular HSP70 induces degranulation and mediator release via phosphorylation of LAT.

Radiotherapy can produce antitumor benefits beyond the local site of irradiation, an immune-based phenomenon known as the abscopal effect, but the mechanisms underlying these benefits are poorly understood. Preclinical studies of ECI301, a mutant derivative of macrophage inhibitory protein-1α, have shown that its administration can improve the antitumor effects of radiotherapy in a manner associated with a tumor-independent abscopal effect. In this article, we report that i.v. administration of ECI301 after intratumoral injection of tumor cell lysates can inhibit tumor growth, not only at the site of injection but also at nontreated sites. Effects of the tumor lysate were further recapitulated by i.v. administration of the alarmins HSP70 or HMGB1, but not HSP60, and combinations of ECI301 + HSP70 were sufficient to inhibit tumor growth. Although injection of ECI301 + HMGB1 did not inhibit tumor growth, we found that administration of a neutralizing HMGB1 antibody neutralized the cooperative effects of ECI301 on tumor irradiation. Moreover, mice genetically deficient in TLR4, an immune pattern receptor that binds alarmins, including HMGB1 and HSP70, did not exhibit antitumor responses to irradiation with ECI301 administration. Although ECI301 was cleared rapidly from peripheral blood, it was found to bind avidly to HSP70 and HMGB1 in vitro. Our results suggest a model in which sequential release of the alarmins HSP70 and HMGB1 from a tumor by irradiation may trap circulating ECI301, thereby licensing or restoring tumor immunosurveillance capabilities of natural killer cells or CD4⁺ and CD8⁺ T cells against tumor cells that may evade irradiation. Cancer Res; 74(18); 5070–8. ©2014 AACR.