Miho Tokumasu

Background and Purpose: Ascochlorin (ASC) is an antiviral antibiotic from the fermented broth of Ascochyta viciae which exerts an inhibitory effect to cancers. Its impact on immune cells has not been examined. In this study, we obtained ASC derivatives with less cytotoxicity and determined whether they affected T cells, indicating possible immune‐mediated antitumour effects. Experimental Approach: Newly synthesised ASC derivatives were screened for inhibitory effects on T‐cell antigen receptor (TCR)‐stimulated proliferative responses using murine CD4 ⁺ and CD8 ⁺ T cells. Two compounds were identified that exhibited >10‐fold less toxicity compared with ASC. N184, the less toxic of the two, was analysed for its in vivo antitumour effects, and in vitro effects on CD8 ⁺ T‐cell proliferation, survival, cytokine production and exhaustion, using microscopy, qPCR and flow cytometry. Key Results: N184 induced limited IL‐9 production in CD8 ⁺ T cells following TCR stimulation, thereby improving cell survival. It also enhanced cytokine production in the late phase of proliferation and suppressed the induction of exhaustion. N184 suppressed tumour growth in mice in a CD8 ⁺ T cell‐dependent manner. The effect was partially prevented by an IL‐9‐neutralising antibody. Conclusion and Implications: N184 induces differentiation of IL‐9‐producing CD8 ⁺ T cells in vitro and elicits antitumour immunity in an IL‐9‐dependent manner.

Fasting is known to alter the circulation dynamics of immune cells, including T cells, by shifting them from peripheral tissues to the bone marrow (BM), where they enter a quiescent state to avoid starvation stress and acquire apoptosis resistance through upregulation of BCL2. Upon refeeding, these T cells exit the BM and return to circulation. In solid tumors, fasting-refeeding not only affects the trafficking of CD8+ T cells between tumors and their draining lymph nodes (dLNs); but also modulates the antitumor immune response. In this study, we investigated how metformin’s antitumor responses are affected by repeated fasting-refeeding cycles. Metformin administration combined with weekly 48-hour fasting showed a synergistic antitumor effect, which was abolished by in vivo depletion of CD8+ T cells. Immunohistofluorescence staining showed that fasting reduced CD8+ T cells in tumors and dLNs while increasing their presence in the BM; refeeding reversed this distribution. Refeeding also increased the expression of Ifng, Gzmb, Tnf, and Tbx21 in tumors. Likewise, Cxcr6, Cxcl16, and Vcam1 expression levels were elevated only upon refeeding. Notably, CXCR6 was exclusively expressed on CD62L− effector memory T cells (TEM). The antitumor effect induced by the combinational therapy was abolished by administration of an anti-VCAM-1 neutralizing antibody. Our findings demonstrate that combining metformin with fasting exerts a synergistic antitumor effect by recruiting CD8+ T cells—relocated to the BM during fasting—back to the tumor during refeeding, facilitated by enhanced VCAM-1 expression on normalized tumor vasculature.

NF-κB mediates transcriptional regulation crucial to many biological functions, and elevated NF-κB activity leads to autoimmune and inflammatory diseases, as well as cancer. Since highly aggressive breast cancers have few therapeutic molecular targets, clarification of key molecular mechanisms of NF-κB signaling would facilitate the development of more effective therapy. In this report, we show that Tob, a member of the Tob/BTG family of antiproliferative proteins, acts as a negative regulator of the NF-κB signal in breast cancer. Studies with 35 human breast cancer cell lines reveal that Tob expression is negatively correlated with NF-κB activity. Analysis of The Cancer Genome Atlas (TCGA) database of clinical samples reveals an inverse correlation between Tob expression and NF-κB activity. Tob knockdown in human breast cancer cells promoted overactivation of NF-κB upon TNF-α treatment, whereas overexpression of Tob inhibited TNF-α stimulation-dependent NF-κB activation. Mechanistically, Tob associates with the TNF receptor complex I and consequently inhibits RIPK1 polyubiquitylation, leading to possible prevention of overwhelming activation of NF-κB.

Tumor blood vessels are highly leaky in structure and have poor blood perfusion, which hampers infiltration and function of CD8T cells within tumor. Normalizing tumor vessels is thus thought to be important in promoting the flux of immune T cells and enhancing ant-tumor immunity. However, how tumor vasculature is normalized is poorly understood. Metformin (Met) combined with ant-PD-1 therapy is known to stimulate proliferation of and to produce large amounts of IFNγ from tumor-infiltrating CD8T lymphocytes (CD8TILs). We found that the combination therapy promotes the pericyte coverage of tumor vascular endothelial cells (ECs) to improve blood perfusion and that it suppresses the hyperpermeability through the increase of VE-cadherin. Peripheral node addressin(PNAd) and vascular cell adhesion molecule (VCAM)-1, both implicated to promote tumor infiltration of CD8T cells, were also increased. Importantly, tumor vessel normalization, characterized as the reduced 70-kDa dextran leakage and the enhancement of VE-cadherin and VCAM-1, were canceled by anti-CD8 Ab or anti-IFNγ Ab injection to mice. The increased CD8TILs were also abrogated by anti-IFNγ Ab injection. In vascular ECs, flow cytometry analysis revealed that pSTAT1 expression was found to be associated with VE-cadherin expression. Moreover, in vitro treatment with Met and IFNγ enhanced VE-cadherin and VCAM-1 on human umbilical vein endothelial cells (HUVECs). The Kaplan–Meier method revealed a correlation of VE-cadherin or VCAM-1 levels with overall survival in patients treated with immune checkpoint inhibitors. These data indicate that IFNγ-mediated cross talk of CD8TILs with tumor vessels is important for creating a better tumor microenvironment and maintaining sustained antitumor immunity.

NF-κB mediates transcriptional regulation crucial to many biological functions, and elevated NF-κB activity leads to autoimmune and inflammatory diseases, as well as cancer. Since highly aggressive breast cancers have few therapeutic molecular targets, clarification of key molecular mechanisms of NF-κB signaling would facilitate development of more effective therapy. In this report, we show that Tob, a member of the Tob/BTG family of antiproliferative proteins, acts as a negative regulator of the NF-κB signal in breast cancer. Studies with 35 human breast cancer cell lines reveal that Tob expression is negatively correlated with NF-κB activity. Analysis of The Cancer Genome Atlas (TCGA) database of clinical samples reveals an inverse correlation between Tob expression and NF-κB activity. Tob2, another member of the Tob/BTG family, shows no such negative correlations. Furthermore, in TNF-α treated cells, Tob associates with TNF receptor complex I to suppress polyubiquitylation of RIPK1, which results in repression of NF-kB activity. Therefore, Tob functions as a negative regulator of the NF-κB pathway and may serve as a therapeutic target for aggressive breast cancer.

Prostaglandin E2 (PGE2), a product of the cyclooxygenase (COX) pathway, is produced by tumors and surrounding stromal cells. It stimulates tumor progression, promotes angiogenesis and suppresses the anti-tumor response. Pharmacological inhibition of PGE2 synthesis has been shown to suppress tumor initiation and growth in vivo. In the current study, we demonstrated that the growth of the Ptgs2-deficient 3LL lung adenocarcinoma cell line was down-regulated in vivo through natural killer (NK) cell activation and a reduction in the population of polymorphonuclear leukocyte-myeloid-derived suppressor cells (PMN-MDSCs) and tumor-associated macrophages (TAMs). On the basis of these results, the therapeutic effect of ONO-AE3-208 (EP4i), an inhibitor of EP4 (a PGE2 receptor), combined with anti-PD-1 antibody was evaluated. EP4i, but not anti-PD-1 antibody, decreased tumor metabolism including glycolysis, fatty acid oxidation and oxidative phosphorylation. EP4i induced IFNγ production from only NK cells (not from T cells) and a shift from M2-like to M1-like macrophages in TAMs. These effects were further enhanced by anti-PD-1 antibody treatment. Although CD8 T-cell infiltration was increased, IFNγ production was not significantly altered, even with combination therapy. Tumor hypoxia was ameliorated by either EP4i or anti-PD-1 antibody treatment, which was further affected by the combination. Normalization of tumor vessels was significant only for the combination therapy. The results indicated a novel effect of EP4i for the metabolic reprogramming of tumors and revealed unique features of EP4i that can synergize with anti-PD-1 antibody to promote IFNγ production by NK cells, polarize TAMs into the M1 phenotype, and reduce hypoxia through normalization of the tumor vasculature.