Sana Hibino

The tumor suppressor microRNA-29c is downregulated and restored by celecoxib in human gastric cancer cells.

MicroRNAs (miRNAs) are small noncoding RNAs that function as endogenous silencers of target genes and play critical roles during carcinogenesis. The selective cyclooxygenase‐2 (COX‐2) inhibitor celecoxib has been highlighted as a potential drug for treatment of gastrointestinal tumors. The aim of this study was to investigate the role of miRNAs in gastric carcinogenesis and the feasibility of a new therapeutic approach for gastric cancer. miRNA expression profiles were examined in 53 gastric tumors including gastric adenomas (atypical epithelia), early gastric cancers and advanced gastric cancers and in gastric cancer cells treated with celecoxib. miRNA microarray analysis revealed that miR‐29c was significantly downregulated in gastric cancer tissues relative to nontumor gastric mucosae. miR‐29c was significantly activated by celecoxib in gastric cancer cells. Downregulation of miR‐29c was associated with progression of gastric cancer and was more prominent in advanced gastric cancers than in gastric adenomas and early gastric cancer. In addition, expression of the oncogene Mcl‐1, a target of miR‐29c, was significantly increased in gastric cancer tissues relative to nontumor gastric mucosae. Activation of miR‐29c by celecoxib induced suppression of Mcl‐1 and apoptosis in gastric cancer cells. These results suggest that downregulation of the tumor suppressor miR‐29c plays critical roles in the progression of gastric cancer. Selective COX‐2 inhibitors may have clinical promise for the treatment of gastric cancer via restoration of miR‐29c.

Inhibitors of enhancer of zeste homolog 2 (EZH2) activate tumor-suppressor microRNAs in human cancer cells

Enhancer of zeste homolog 2 (EZH2) enhances tumorigenesis and is commonly overexpressed in several types of cancer. To investigate the anticancer effects of EZH2 inhibitors, microRNA (miRNA) expression profiles were examined in gastric and liver cancer cells treated with suberoylanilide hydroxamic acid (SAHA) and 3-deazaneplanocin A (DZNep). We confirmed that SAHA and DZNep suppressed EZH2 expression in AGS and HepG2 cells and inhibited their proliferation. The results of microarray analyses demonstrated that miR-1246 was commonly upregulated in cancer cells by treatment with SAHA and DZNep. MiR-302a and miR-4448 were markedly upregulated by treatment with SAHA and DZNep, respectively. DYRK1A, CDK2, BMI-1 and Girdin, which are targets of miR-1246, miR-302a and miR-4448, were suppressed by treatment with SAHA and DZNep, leading to apoptosis, cell cycle arrest and reduced migration of AGS and HepG2 cells. ChIP assay revealed that SAHA and DZNep inhibited the binding of EZH2 to the promoter regions of miR-1246, miR-302a and miR-4448. These findings suggest that EZH2 inhibitors such as SAHA and DZNep exert multiple anticancer effects through activation of tumor-suppressor miRNAs.

Alterations of epigenetics and microRNA in hepatocellular carcinoma.

Studies have shown that alterations of epigenetics and microRNA (miRNA) play critical roles in the initiation and progression of hepatocellular carcinoma (HCC). Epigenetic silencing of tumor suppressor genes in HCC is generally mediated by DNA hypermethylation of CpG island promoters and histone modifications such as histone deacetylation, methylation of histone H3 lysine 9 (H3K9) and tri‐methylation of H3K27. Chromatin‐modifying drugs such as DNA methylation inhibitors and histone deacetylase inhibitors have shown clinical promise for cancer therapy. miRNA are small non‐coding RNA that regulate expression of various target genes. Specific miRNA are aberrantly expressed and play roles as tumor suppressors or oncogenes during hepatocarcinogenesis. We and other groups have demonstrated that important tumor suppressor miRNA are silenced by epigenetic alterations, resulting in activation of target oncogenes in human malignancies including HCC. Restoring the expression of tumor suppressor miRNA by inhibitors of DNA methylation and histone deacetylase may be a promising therapeutic strategy for HCC.

Regulation of regulatory T cells: epigenetics and plasticity.

Regulatory T (Treg) cells, as central mediators of immune suppression, play crucial roles in many aspects of immune systems physiology and pathophysiology. The transcription factor Foxp3 has been characterized as a master gene of Tregs. Yet Treg cells possess a distinct pattern of gene expression, including upregulation of immune-suppressive genes and silencing of inflammatory cytokine genes. Recent studies have revealed the molecular mechanisms that establish and maintain such gene regulation in Treg cells. This review discusses recent progress in our understanding of molecular features of Treg cells, with particular attention to Treg-cell lineage commitment and stability.

Loss of Sprouty4 in T cells ameliorates experimental autoimmune encephalomyelitis in mice by negatively regulating IL-1β receptor expression

Th17 cells, which have been implicated in autoimmune diseases, require IL-6 and TGF-β for early differentiation. To gain pathogenicity, however, Th17 cells require IL-1β and IL-23. The underlying mechanism by which these confer pathogenicity is not well understood. Here we show that Sprouty4, an inhibitor of the PLCγ-ERK pathway, critically regulates inflammatory Th17 (iTh17) cell differentiation. Sprouty4-deficient mice, as well as mice adoptively transferred with Sprouty4-deficient T cells, were resistant to experimental autoimmune encephalitis (EAE) and showed decreased Th17 cell generation in vivo. In vitro, Sprouty4 deficiency did not severely affect TGF-β/IL-6-induced Th17 cell generation but strongly impaired Th17 differentiation induced by IL-1/IL-6/IL-23. Analysis of Th17-related gene expression revealed that Sprouty4-deficient Th17 cells expressed lower levels of IL-1R1 and IL-23R, while RORγt levels were similar. Consistently, overexpression of Sprouty4 or pharmacological inhibition of ERK upregulated IL-1R1 expression in primary T cells. Thus, Sprouty4 and ERK play a critical role in developing iTh17 cells in Th17 cell-driven autoimmune diseases.

Nr4a Receptors Regulate Development and Death of Labile Treg Precursors to Prevent Generation of Pathogenic Self-Reactive Cells

Regulatory T (Treg) cells develop from a self-reactive, CD4-single positive (CD4SP) precursor cell pool. Thus, Treg-fated developing thymocytes are expected to possess the potential to generate pathogenic self-reactive cells. However, no such pathogenic conversion has been observed, indicating mechanisms of defense to prevent such a deleterious event. Here, we show that, after the initial developmental phase, the Nr4a family of nuclear receptors promotes the development of Treg cells by cooperating with other Treg cell developmental machineries, as well as by forming a reinforcing loop with Foxp3. Nr4a-deficient Treg-fated thymocytes survive and can elicit autoimmunity, highlighting their roles in elimination of developing Treg precursors that fail to complete their development. Our findings reveal that the defective development of Treg-fated thymocytes is a potential route for the generation of pathogenic self-reactive cells, which is normally suppressed by Nr4a factors at both developmental and cell death levels.

Generation and application of human induced-stem cell memory T cells for adoptive immunotherapy

Adoptive T‐cell therapy is an effective strategy for cancer immunotherapy. However, infused T cells frequently become functionally exhausted, and consequently offer a poor prognosis after transplantation into patients. Adoptive transfer of tumor antigen‐specific stem cell memory T (TSCM) cells is expected to overcome this shortcoming as TSCM cells are close to naïve T cells, but are also highly proliferative, long‐lived, and produce a large number of effector T cells in response to antigen stimulation. We previously reported that activated effector T cells can be converted into TSCM‐like cells (iTSCM) by coculturing with OP9 cells expressing Notch ligand, Delta‐like 1 (OP9‐hDLL1). Here we show the methodological parameters of human CD8+ iTSCM cell generation and their application to adoptive cancer immunotherapy. Regardless of the stimulation by anti‐CD3/CD28 antibodies or by antigen‐presenting cells, human iTSCM cells were more efficiently induced from central memory type T cells than from effector memory T cells. During the induction phase by coculture with OP9‐hDLL1 cells, interleukin (IL)‐7 and IL‐15 (but not IL‐2 or IL‐21) could efficiently generate iTSCM cells. Epstein–Barr virus‐specific iTSCM cells showed much stronger antitumor potentials than conventionally activated T cells in humanized Epstein–Barr virus transformed‐tumor model mice. Thus, adoptive T‐cell therapy with iTSCM offers a promising therapeutic strategy for cancer immunotherapy.

Inhibition of Nr4a Receptors Enhances Antitumor Immunity by Breaking Treg-Mediated Immune Tolerance

Enhanced infiltration of regulatory T cells (Treg) into tumor tissue is detrimental to patients with cancer and is closely associated with poor prognosis as they create an immunosuppressive state that suppresses antitumor immune responses. Therefore, breaking Treg-mediated immune tolerance is important when considering cancer immunotherapy. Here, we show that the Nr4a nuclear receptors, key transcription factors maintaining Treg genetic programs, contribute to Treg-mediated suppression of antitumor immunity in the tumor microenvironment. Mice lacking Nr4a1 and Nr4a2 genes specifically in Tregs showed resistance to tumor growth in transplantation models without exhibiting any severe systemic autoimmunity. The chemotherapeutic agent camptothecin and a common cyclooxygenase-2 inhibitor were found to inhibit transcriptional activity and induction of Nr4a factors, and they synergistically exerted antitumor effects. Genetic inactivation or pharmacologic inhibition of Nr4a factors unleashed effector activities of CD8+ cytotoxic T cells and evoked potent antitumor immune responses. These findings demonstrate that inactivation of Nr4a in Tregs breaks immune tolerance toward cancer, and pharmacologic modulation of Nr4a activity may be a novel cancer treatment strategy targeting the immunosuppressive tumor microenvironment.Significance: This study reveals the role of Nr4a transcription factors in Treg-mediated tolerance to antitumor immunity, with possible therapeutic implications for developing effective anticancer therapies. Cancer Res; 78(11); 3027-40. ©2018 AACR.

Tu1900 MiR-1246 and Mir-302 Are Novel Targets of Epigenetic Therapy With Dznep and SAHA in Human Cancer Cells

Introduction Epigenetic therapy with DNA methylation inhibitors and histone deacetylase (HDAC) inhibitors holds clinical promise for the treatment of human malignancies. MicroRNAs (miRNAs) are small non-coding RNAs that function as endogenous silencers of various target genes and play critical roles in cancer. We have proposed that epigenetic activation of tumor suppressor miRNAs can be a novel therapeutic approach for human cancers (Cancer Cell 9: 435, 2006). Recently, 3-deazaneplanocin A (DZNep) was discovered to inhibit a polycomb group protein, enhancer of zeste homologue 2 (EZH2), which has activity for trimethylation of histone H3K27. To investigate the molecular mechanisms underlying the effect of epigenetic therapy with DZNep and the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) against human cancer, miRNA expression profiles were analyzed. Materials and Methods AGS human gastric cancer cells and HepG2 human liver cancer cells were treated with DZNep and SAHA. MiRNA expression profiles were analyzed by microarray analysis and TaqMan quantitative RT-PCR. Cell proliferation was analyzed by cell counting, and target genes of miRNAs were analyzed by Western blotting. Levels of apoptosis and the cell cycle were analyzed by flow cytometry. Results The results of microarray and quantitative RT-PCR analyses revealed that miR-1246 was up-regulated by treatment with DZNep in both AGS and HepG2 cells, and that miR-302 was up-regulated by treatment with SAHA in AGS cells. Treatment with DZNep or SAHA reduced the cell proliferation activity of both AGS and HepG2 cells, and this reduction was more marked when both drugs were used in combination. The anti-apoptotic factor DYRK1A, which was recently identified as one of the targets of miR-1246, was significantly suppressed by treatment with DZNep and SAHA, resulting in apoptosis of AGS andHepG2 cells. In addition, treatment of AGS cells with DZNep and SAHA suppressed CDK2 and BMI-1, which were recently identified as the targets of miR-302, inducing cell cycle (G1/S) arrest of AGS cells. Conclusions These findings suggest that DZNep is a promising drug for the treatment of gastric and liver cancers via activation of miR-1246. DZNep and SAHA may synergistically suppress the proliferation of gastric cancer cells via activation of miR-1246 and miR-302. MiR-1246 and miR-302 are novel targets of epigenetic therapy with DZNep and SAHA in human cancer cells.