Wachiko Nakata

Apoptosis signal-regulating kinase 1 and cyclin D1 compose a positive feedback loop contributing to tumor growth in gastric cancer.

Mitogen-activated protein kinase (MAPK) pathways regulate multiple cellular functions and are highly active in many types of human cancers. Apoptosis signal-regulating kinase 1 (ASK1) is an upstream MAPK involved in apoptosis, inflammation, and carcinogenesis. This study investigated the role of ASK1 in the development of gastric cancer. In human gastric cancer specimens, we observed increased ASK1 expression, compared to nontumor epithelium. Using a chemically induced murine gastric tumorigenesis model, we observed increased tumor ASK1 expression, and ASK1 knockout mice had both fewer and smaller tumors than wild-type (WT) mice. ASK1 siRNA inhibited cell proliferation through the accumulation of cells in G1 phase of the cell cycle, and reduced cyclin D1 expression in gastric cancer cells, whereas these effects were uncommon in other cancer cells. ASK1 overexpression induced the transcription of cyclin D1, through AP-1 activation, and ASK1 levels were regulated by cyclin D1, via the Rb–E2F pathway. Exogenous ASK1 induced cyclin D1 expression, followed by elevated expression of endogenous ASK1. These results indicate an autoregulatory mechanism of ASK1 in the development of gastric cancer. Targeting this positive feedback loop, ASK1 may present a potential therapeutic target for the treatment of advanced gastric cancer.

Apoptosis Signal-Regulating Kinase 1 Inhibits Hepatocarcinogenesis by Controlling the Tumor-Suppressing Function of Stress-Activated Mitogen-Activated Protein Kinase

The stress‐activated mitogen‐activated protein kinases (MAPKs), c‐Jun NH2‐terminal kinase (JNK), and p38 have been implicated in hepatocarcinogenesis. Although the many interrelated functions of JNK and p38 are precisely regulated by upstream signaling molecules, little is known about upstream regulators. We investigated the role of apoptosis signal‐regulating kinase 1 (ASK1), a major player in the regulation of JNK and p38 activities, in hepatocarcinogenesis using a mouse hepatocellular carcinoma (HCC) model. ASK1‐deficient (ASK1−/−) and wildtype (WT) mice were treated with diethylnitrosamine on postnatal day 14. Strikingly, after 7 months, approximately three times as many tumors developed in ASK1−/− mice as in WT mice. Although JNK and p38 activation were attenuated in ASK1−/− HCCs relative to WT HCCs, cell proliferation was comparable in HCCs from both types of mice. On the other hand, both cancer cell apoptosis and hyperphosphorylation of BimEL, a proapoptotic Bcl‐2 family member, were suppressed in the ASK1−/− HCCs. ASK1−/− mice showed remarkable resistance to Fas‐induced hepatocyte apoptosis in vivo, probably because of attenuated JNK‐mediated BimEL phosphorylation and mitochondrial apoptotic pathway activation. The reintroduction of ASK1 to ASK1−/− mouse liver using an adenoviral vector restored Fas‐induced hepatocyte death and phosphorylation of JNK and BimEL. Similar findings were obtained in tumor necrosis factor alpha‐induced hepatocyte apoptosis. Furthermore, ASK1 was involved in DNA damage‐induced p21 up‐regulation through a p38 pathway. Conclusion: ASK1 is involved in death receptor‐mediated apoptosis and DNA‐damage response by way of stress‐activated MAPK in the liver, and thus acts as a tumor suppressor in hepatocarcinogenesis. This study provides new insight into the regulation of stress‐ activated MAPK signaling in hepatocarcinogenesis. (HEPATOLOGY 2011;)

Interleukin-6 mediates epithelial-stromal interactions and promotes gastric tumorigenesis.

Interleukin-6 (IL-6) is a pleiotropic cytokine that affects various functions, including tumor development. Although the importance of IL-6 in gastric cancer has been documented in experimental and clinical studies, the mechanism by which IL-6 promotes gastric cancer remains unclear. In this study, we investigated the role of IL-6 in the epithelial–stromal interaction in gastric tumorigenesis. Immunohistochemical analysis of human gastritis, gastric adenoma, and gastric cancer tissues revealed that IL-6 was frequently detected in the stroma. IL-6–positive cells in the stroma showed positive staining for the fibroblast marker α-smooth muscle actin, suggesting that stromal fibroblasts produce IL-6. We compared IL-6 knockout (IL-6−/−) mice with wild-type (WT) mice in a model of gastric tumorigenesis induced by the chemical carcinogen N-methyl-N-nitrosourea. The stromal fibroblasts expressed IL-6 in tumors from WT mice. Gastric tumorigenesis was attenuated in IL-6−/− mice, compared with WT mice. Impaired tumor development in IL-6−/− mice was correlated with the decreased activation of STAT3, a factor associated with gastric cancer cell proliferation. In vitro, when gastric cancer cell line was co-cultured with primary human gastric fibroblast, STAT3–related genes including COX-2 and iNOS were induced in gastric cancer cells and this response was attenuated with neutralizing anti-IL-6 receptor antibody. IL-6 production from fibroblasts was increased when fibroblasts were cultured in the presence of gastric cancer cell–conditioned media. IL-6 production from fibroblasts was suppressed by an interleukin-1 (IL-1) receptor antagonist and siRNA inhibition of IL-1α in the fibroblasts. IL-1α mRNA and protein were increased in fibroblast lysate, suggesting that cell-associated IL-1α in fibroblasts may be involved. Our results suggest the importance of IL-6 mediated stromal-epithelial cell interaction in gastric tumorigenesis.

Role of Interleukin-32 in Helicobacter pylori-Induced Gastric Inflammation

ABSTRACT Helicobacter pylori infection is associated with gastritis and gastric cancer. An H. pylori virulence factor, the cag pathogenicity island (PAI), is related to host cell cytokine induction and gastric inflammation. Since elucidation of the mechanisms of inflammation is important for therapy, the associations between cytokines and inflammatory diseases have been investigated vigorously. Levels of interleukin-32 (IL-32), a recently described inflammatory cytokine, are increased in various inflammatory diseases, such as rheumatoid arthritis and Crohns disease, and in malignancies, including gastric cancer. In this report, we examined IL-32 expression in human gastric disease. We also investigated the function of IL-32 in activation of the inflammatory cytokines in gastritis. IL-32 expression paralleled human gastric tissue pathology, with low IL-32 expression in H. pylori-uninfected gastric mucosa and higher expression levels in gastritis and gastric cancer tissues. H. pylori infection increased IL-32 expression in human gastric epithelial cell lines. H. pylori-induced IL-32 expression was dependent on the bacterial cagPAI genes and on activation of nuclear factor κB (NF-κB). IL-32 expression induced by H. pylori was not detected in the supernatant of AGS cells but was found in the cytosol. Expression of the H. pylori-induced cytokines CXCL1, CXCL2, and IL-8 was decreased in IL-32-knockdown AGS cell lines compared to a control AGS cell line. We also found that NF-κB activation was decreased in H. pylori-infected IL-32-knockdown cells. These results suggest that IL-32 has important functions in the regulation of cytokine expression in H. pylori-infected gastric mucosa.

Apoptosis signal-regulating kinase-1 inhibitor as a potent therapeutic drug for the treatment of gastric cancer.

Aside from the human epidermal growth factor receptor‐2 (HER2)‐targeting agent trastuzumab, molecular targeting therapy for gastric cancer (GC) has not been established. We previously reported that apoptosis signal‐regulating kinase‐1 (ASK1) was upregulated in human GC and that overexpression of ASK1 promoted GC cell proliferation. Here, we investigated the effect of ASK1 inhibitor K811 on GC cells. K811 efficiently prevented cell proliferation in cell lines with high ASK1 expression and in HER2‐overexpressing GC cells. Treatment with K811 reduced sizes of xenograft tumors by downregulating proliferation markers. These results indicate that ASK1 inhibition prevents GC cell growth in vitro and in vivo, suggesting that ASK1 inhibitors can be potent therapeutic drugs for GC.

Therapeutic effect of c-Jun N-terminal kinase inhibition on pancreatic cancer

c‐Jun N‐terminal kinase (JNK) is a member of the mitogen‐activated protein kinase (MAPK) family, and it is reportedly involved in the development of several cancers. However, the role of JNK in pancreatic cancer has not been elucidated. We assessed t he involvement of JNK in the development of pancreatic cancer and investigated the therapeutic effect of JNK inhibitors on this deadly cancer. Small interfering RNAs against JNK or the JNK inhibitor SP600125 were used to examine the role of JNK in cellular proliferation and the cell cycles of pancreatic cancer cell lines. Ptf1acre/+;LSL‐KrasG12D/+;Tgfbr2flox/flox mice were treated with the JNK inhibitor to examine pancreatic histology and survival. The effect of JNK inhibition on tumor angiogenesis was also assessed using cell lines and murine pancreatic cancer specimens. JNK was frequently activated in human and murine pancreatic cancer in vitro and in vivo. Growth of human pancreatic cancer cell lines was suppressed by JNK inhibition through G1 arrest in the cell cycle with decreased cyclin D1 expression. In addition, oncogenic K‐ras expression led to activation of JNK in pancreatic cancer cell lines. Treatment of Ptf1acre/+;LSL‐KrasG12D/+;Tgfbr2flox/flox mice with the JNK inhibitor decreased growth of murine pancreatic cancer and prolonged survival of the mice significantly. Angiogenesis was also decreased by JNK inhibition in vitro and in vivo. In conclusion, activation of JNK promotes development of pancreatic cancer, and JNK may be a potential therapeutic target for pancreatic cancer.

Differential roles of ASK1 and TAK1 in Helicobacter pylori-induced cellular responses

ABSTRACT The mitogen-activated protein kinase (MAPK) signaling pathway regulates various cellular functions, including those induced by Helicobacter pylori. TAK1 is an upstream MAPK kinase kinase (MAP3K) required for H. pylori-induced MAPK and NF-κB activation, but it remains unclear whether other MAP3Ks are involved in H. pylori-induced cellular responses. In this study, we focused on the MAP3K ASK1, which plays a critical role in gastric tumorigenesis. In gastric epithelial cells, H. pylori activates ASK1 in a reactive oxygen species (ROS)- and cag pathogenicity island-dependent manner, and ASK1 regulates sustained JNK activation and apoptosis induced by H. pylori. In contrast, TAK1 regulates H. pylori-mediated early JNK activation and cytokine production. We also found reciprocal regulation between ASK1 and TAK1 in H. pylori-related responses, whereby inhibition of TAK1 or downstream p38 MAPK activates ASK1 through ROS production, and ASK1 suppresses TAK1 and downstream NF-κB activation. We identified ROS/ASK1/JNK as a new signaling pathway induced by H. pylori, which regulates apoptotic cell death. The balance of ASK1-induced apoptosis and TAK1-induced antiapoptotic or inflammatory responses may determine the fate of epithelial cells infected with H. pylori and thus be involved in the pathogenesis of gastritis and gastric cancer.

Promotion of DNA repair by nuclear IKKβ phosphorylation of ATM in response to genotoxic stimuli.

Ataxia-telangiectasia mutated (ATM) is one of the key molecules involved in the cellular response to DNA damage. A portion of activated ATM is exported from the nucleus into the cytoplasm, where it activates the I kappa B kinase/nuclear factor kappa B (IKK/NF-κB) signaling pathway. It has been thought that activated IKKβ, which is a critical kinase for NF-κB activation, generally resides in the cytoplasm and phosphorylates cytoplasmic downstream molecules, such as IκBα. Here, we identified a new role for IKKβ during the response to DNA damage. ATM phosphorylation in response to alkylating agents consisted of two phases: the early phase (up to 3 h) and late phase (after 6 h). A portion of the activated IKKβ generated during the DNA damage response was found to translocate into the nucleus and directly phosphorylate ATM in the late phase. Furthermore, the phosphorylation of ATM by nuclear IKKβ was suggested to promote DNA repair. In parallel, activated IKKβ induced classical NF-κB activation and was involved in anti-apoptosis. Our findings define the function of IKKβ during the response to DNA damage, which promotes cell survival and DNA repair, and maintains cellular homeostasis.

Abstract 2740: The role of JNK in the development of pancreatic cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC C-Jun NH2-terminal kinase (JNK) is a member of mitogen-activated protein kinase (MAPK) family, and it is known to regulate a variety of cellular activities including apoptosis, survival, differentiation, and proliferation. Recently, it has been suggested that JNK is involved in the development of several cancers, but the role of JNK in pancreatic cancer is not fully elucidated. In this study, we examined the role of JNK in the development of pancreatic cancer and evaluated the therapeutic effect of JNK inhibition on pancreatic cancer. In immunohistochemical staining, JNK activation was observed in human pancreatic cancer specimens. Growth of pancreatic cancer cell lines was inhibited by treatment with JNK inhibitor SP600125 or by transfection of siRNAs against JNK1 or JNK2. Expression of cyclin D1 in pancreatic cancer cells was decreased by JNK inhibition, and cell cycle analysis showed accumulation of cells in G0/G1 phase by JNK1 and JNK2 inhibition. Induction of oncogenic Ras into pancreatic cancer cells promoted JNK activation, and KRAS knockdown by siRNA decreased phosphorylation of JNK. Cyclin D1 expression was shown to be regulated through JNK activation by luciferase assay and real-time RT-PCR. Pancreas-specific KrasG12D expression and type II TGFβreceptor knockout mice (KrasG12D+Tgfbr2KO mice) was used as a mouse model of pancreatic cancer, and pancreatic cancer developed in KrasG12D+Tgfbr2KO mice showed higher activation of JNK than PanIN tissue of KrasG12D mice or normal pancreas of wild-type mice. Treating KrasG12D+Tgfbr2KO mice with JNK inhibitor for 4 weeks led to less progression of pancreatic cancer, and immunohistochemical staining showed reduced expression of phosphorylated JNK, c-jun, cyclin D1 and PCNA in the pancreatic cancer tissues compared to control vehicle. The survival time of KrasG12D+Tgfbr2KO mice was significantly prolonged by SP600125 treatment. Decreased number of blood vessels was observed in pancreatic cancer tissue of KrasG12D+Tgfbr2KO mice treated by SP600125. Secretion of angiogenic cytokines from pancreatic cancer cell lines was decreased by JNK inhibition, and angiogenesis by HUVEC was inhibited by incubating in the conditioned medium of pancreatic cancer cells treated by SP600125 or transfected with siRNAs against JNK1 or JNK2, indicating the effect of JNK inhibition on tumor angiogenesis. These data indicate that oncogenic K-ras activates JNK JNK is involved in the development of pancreatic cancer through the regulation of cell cycle and tumor angiogenesis. Inhibiting JNK may be a potential therapy for pancreatic cancer. Citation Format: Ryota Takahashi, Yoshihiro Hirata, Kosuke Sakitani, Wachiko Nakata, Hiroto Kinoshita, Yoku Hayakawa, Hayato Nakagawa, Hideaki Ijichi, Shin Maeda, Kazuhiko Koike. The role of JNK in the development of pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2740. doi:10.1158/1538-7445.AM2013-2740

Su1658 The Role of Interleukin-32 in Helicobacter pylori Induced Gastric Inflammation

Introduction: Transactivation of the EGFR involves a triple membrane passing signal (TMPS) via ADAM17 and HB-EGF in gastric epithelial cells is of relevance to gastric carcinogenesis. H. pylori-induced ADAM17 transcript levels are significantly inhibited by EGCG, a natural product from green tea, in MKN-28 human gastric epithelial cells. The aim of this study was to investigate if H. pylori-induced HB-EGF in MKN-28 gastric epithelial cells and H. pylori-inducedAdam17 andHb-egf in immortalizedmouse gastric epithelial cells (MGEC)were inhibited by EGCG.Methods: After optimal co-culture time with H. pylori (NCTC11637, cag PAI+) at 12 hrs was identified, MKN-28 or MGEC gastric epithelial cells were pre-incubated for 1 hr with EGCG (1-100 μM) (concentrations which did not affect bacterial or cell viability) before co-culture withH. pylori. At 12 hrs post-infection whenmaximum expression ofHB-EGF, Adam17 andHb-egfwas evident, RNA was extracted form both cells and evaluated by Northern blotting. Results: HB-EGF transcript levels in MKN-28 cells were significantly reduced (p<0.05, n=3) by EGCG compared to untreated H. pylori controls at a concentration of 100 μM EGCG at 12 hrs post-infection. EGCG (50 μM) significantly inhibited (p<0.05, n = 3) H. pylori-induced Adam17 and Hb-egf transcripts in MGEC cells at 12 hrs postinfection. Conclusions: H. pylori-induced HB-EGF in MKN-28 cells and Adam17 with Hbegf in MGEC cells are significantly inhibited by EGCG. Green tea consumption as an inhibitor for H. pylori-induced gastric cancer, via the TMPS pathway, might have a role in prevention of gastric carcinogenesis.