Hideya Onishi

Lipopolysaccharide (LPS) increases the invasive ability of pancreatic cancer cells through the TLR4/MyD88 signaling pathway†

Inflammation plays a multifaceted role in cancer progression, and NF‐κB is one of the key factors connecting inflammation with cancer progression. We have shown that lipopolysaccharide (LPS) promotes NF‐κB activation in colon cancer cells and pancreatic cancer cells. However, it is unclear why inflammatory stimuli can induce NF‐κB activation in cancer cells.

Hedgehog signaling pathway as a therapeutic target in various types of cancer

Hedgehog (Hh) signaling is an important factor in growth and patterning during embryonic development. A mutation in Patched, Smoothened or Gli1, which regulate the Hh signaling pathway, might lead to the onset of glioblastoma, basal cell carcinoma, medulloblastoma and rhabdomyosarcoma. Recently, Hh signaling has been reported to be activated in a ligand‐dependent manner, contributing to carcinogenesis and cancer progression. Hedgehog signaling is reactivated in various types of cancer, and this contributes to cancer progression by facilitating proliferation, invasion and cell survival. Moreover, Hh signaling is associated with several other signaling pathways that contribute to cancer progression. These observations indicate that controlling Hh signaling might become a target for novel molecular targeting therapy. (Cancer Sci 2011; 102: 1756–1760)

Hypoxia activates the hedgehog signaling pathway in a ligand-independent manner by upregulation of Smo transcription in pancreatic cancer

The hedgehog (Hh) signaling pathway is activated in various types of cancer including pancreatic ductal adenocarcinoma. It has been shown that extremely low oxygen tension (below 1% O2) is found in tumor tissue including pancreatic ductal adenocarcinoma cells (PDAC) and increases the invasiveness of PDAC. To investigate the contribution of the Hh pathway to hypoxia‐induced invasiveness, we examined how hypoxia affects Hh pathway activation and the invasiveness of PDAC. In the present study, three human PDAC lines were cultured under normoxic (20% O2) or hypoxic (1% O2) conditions. Hypoxia upregulated the transcription of Sonic hedgehog (Shh), Smoothened (Smo), Gli1 and matrix metalloproteinase9 (MMP9) and increased the invasiveness of PDAC. Significantly, neither the addition of recombinant Shh (rhShh) nor the silencing of Shh affected the transcription of these genes and the invasiveness of PDAC. On the other hand, silencing of Smo decreased the transcription of Gli1 and MMP9 and PDAC invasiveness. Silencing of Gli1 or MMP9 decreased PDAC invasiveness. These results suggest that hypoxia activates the Hh pathway of PDAC by increasing the transcription of Smo in a ligand‐independent manner and increases PDAC invasiveness. (Cancer Sci 2011; 102: 1144–1150)

VEGFR2 is selectively expressed by FOXP3high CD4+ Treg

CD25+ FOXP3+CD4+ T cells (Treg) have been considered to play an important role in immune tolerance against several tumor antigens. It has also been indicated that high‐level expression of FOXP3 (FOXP3high) is sufficient to confer suppressive activity to normal non‐Treg. Here, we showed for the first time that vascular endothelial growth factor receptor 2 (VEGFR2) is selectively expressed by FOXP3high but not FOXP3low Treg. Such VEGFR2+ Treg exist in several tissues including PBMC and malignant effusion‐derived lymphocytes. In conclusion, VEGFR2 may be a novel target for controlling Treg with highly suppressive function.

Hedgehog signaling pathway mediates the progression of non‐invasive breast cancer to invasive breast cancer

The purpose of this study is to clarify the contribution of the Hedgehog signaling pathway (Hh pathway) to the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). A total of 149 surgically resected mammary disease specimens and 12 sentinel lymph nodes with micro‐metastasis (Ly‐met) were studied. The degree of Hh pathway activation was estimated from the Gli1 nuclear staining ratio (%Gli1 nuclear translocation) in cancer cells. The invasiveness of breast cancer cells was determined using Matrigel assays. A serial increase of %Gli1 nuclear translocation to IDC from non‐neoplastic diseases was confirmed. In tumor specimens, %Gli1 nuclear translocation correlated with the invasiveness of each type of mammary disease and also correlated with invasion‐related histopathological parameters. The %Gli1 nuclear translocation in lymph nodes with micro‐metastasis was similar to that in primary sites and higher than that in DCIS with microinvasion and DCIS. Blockade of the Hh pathway decreased the invasiveness of breast cancer cells. In IDC, %Gli1 nuclear translocation correlated with the expression of estrogen receptor‐α. Estrogen increased %Gli1 nuclear translocation and the invasiveness of estrogen receptor‐α‐positive cells. The Hh pathway mediates progression from a non‐invasive phenotype to an invasive phenotype and %Gli1 nuclear translocation may be useful as a predictive marker for evaluating the ability of invasiveness. (Cancer Sci 2011; 102: 373–381)

Dendritic cell‐based combined immunotherapy with autologous tumor‐pulsed dendritic cell vaccine and activated T cells for cancer patients: rationale, current progress, and perspectives

Effective adoptive cancer immunotherapy depends on an ability to generate tumor-antigen-presenting cells and tumor-reactive effector lymphocytes and to deliver these effector cells to the tumor. Dendritic cells (DCs) are the most potent antigen-presenting cells, capable of sensitizing T cells to new and recall antigens. Many studies have shown that tumors express unique proteins that can be loaded on DCs to lrigger an immune response. The current experimental and clinical statuses of adoptive transfer of tumor antigen-pulsed DCs and vaccine-primed activated T cells are summarized herein. Clinical trials of antigen-pulsed DCs have been conducted in patients with various types of cancer, including non-Hodgkin lymphoma, multiple myeloma, prostate cancer, renal cell carcinoma, malignant melanoma, colorectal cancer, and non-small cell lung cancer. These studies have shown that antigen-loaded DC vaccination is safe and promising for the treatment of cancer. In addition, tumor vaccine-primed T cells have been shown to induce antitumor activity in vivo. Several clinical studies are being conducted on the use of vaccine-primed T cells such as tumor-drainage lymph node. It is reasonable to consider using both tumor antigen-pulsed DCs and vaccine-primed lymphocytes as adjuvants. We are now investigating the use of autologous whole tumor antigen-pulsed DCs and the DC vaccine-primed activated lymphocytes in patients with multiple metastasis of solid tumors.

Hedgehog signaling pathway as a new therapeutic target in pancreatic cancer

Pancreatic cancer is one of the most aggressive and difficult cancers to treat. Despite numerous research efforts, limited success has been achieved in the therapeutic management of patients with this disease. In the current review, we focus on one component of morphogenesis signaling, Hedgehog (Hh), with the aim of developing novel, effective therapies for the treatment of pancreatic cancer. Hh signaling contributes to the induction of a malignant phenotype in pancreatic cancer and is responsible for maintaining pancreatic cancer stem cells. In addition, we propose a novel concept linking Hh signaling and tumor hypoxic conditions, and discuss the effects of Hh inhibitors in clinical trials. The Hh signaling pathway may represent a potential therapeutic target for patients with refractory pancreatic cancer.

Nuclear factor kappaB-activated monocytes contribute to pancreatic cancer progression through the production of Shh.

Recently, it was reported that Hh signaling is activated in tumor stromal cells but not in tumor cells themselves and that stromal cells may play a role in the proliferation of cancer cells. This suggests the possibility that stromal cells have an important role in the proliferation of tumor cells that may be mediated through Hh signaling. In this report, we present for the first time that inflammation-stimulated monocytes produce Shh through activation of the NF-κB signaling pathway, and that the Shh produced promotes the proliferation of pancreatic cancer cells in a paracrine manner through Hh signaling.

TrkB/BDNF signaling pathway is a potential therapeutic target for pulmonary large cell neuroendocrine carcinoma

Tropomyosin-related kinase B (TrkB) plays an important role in tumor progression in various kinds of cancers; however, little is known about biological significance of TrkB in human lung cancer, especially large cell neuroendocrine carcinoma (LCNEC). We hereby investigated the expressions of TrkB and its ligand brain-derived neurotrophic factor (BDNF) in clinical specimens and their influences on phenotypes of invasiveness and tumorigenicity for LCNEC. The expressions of TrkB and BDNF analyzed by immunohistochemistry for patients samples with lung cancer (n=104) were significantly higher in neuroendocrine tumor (NET) compared with non-NET. In particular, LCNEC, a subtype of NET, exhibited significantly higher TrkB and BDNF expressions than another NET type: small cell lung cancer (SCLC), and a significant correlation between TrkB and BDNF expressions was noted in LCNEC but not in SCLC. In vitro assay, exogenous BDNF addition enhanced the invasion into matrigels of LCNEC cells, whereas inhibition of TrkB or BDNF suppressed matrix metalloproteinase-2 and -9 activities and the invasiveness. Exogenous BDNF also increased anchor-independent colony formation on soft agar gels for LCNEC, while inhibition of TrkB or BDNF suppressed the anchorage-independency. In vivo experiments, implanted LCNEC cells pretreated with TrkB-siRNA developed no subcutaneous tumor in all six nude mice, although those with control-siRNA formed tumors in four of six nude mice. In conclusion, BDNF/TrkB signal is involved in malignant progression of invasiveness and tumorigenicity for LCNEC, and may be a potential target for LCNEC without standard therapy.

Interferon-gamma suppresses transforming growth factor-beta-induced invasion of gastric carcinoma cells through cross-talk of Smad pathway in a three-dimensional culture model.

We reconstituted a three-dimensional gastric carcinoma model similar to invasive gastric carcinoma tissue. This model consists of a human gastric carcinoma cell line, GCTM-1, a human fibroblast cell line, TIG-1-20, and transforming growth factor-β (TGF-β)-containing type I collagen gel. Using this model, we were able to observe the growth of the two cell types, especially carcinoma cell invasive growth, in real time for more than 30 days. TGF-β and TIG-1-20 were essential for GCTM-1 invasive growth and proliferation, respectively. TGF-β induced the enhanced expression of matrix metalloproteinase 9 (MMP9) and urokinase-type plasminogen activator (uPA) in GCTM-1 at both the protein and enzymatic activity levels. The TGF-β-induced invasion of GCTM-1 was inhibited by MMP9- or uPA-antisense (AS) oligonucleotide transfection to GCTM-1. When exogenous interferon-γ (IFN-γ) was added to this model, TGF-β-dependent GCTM-1 invasion was significantly inhibited, concomitant with the decreased expression of MMP9 and uPA. The intracellular signal transduction of Smad was examined to analyse the mechanism of the inhibitory effect of IFN-γ. TGF-β accelerated the phosphorylation of Smad2/3 and nuclear translocation of the Smad2/3–Smad4 complex in GCTM-1, but these TGF-β-induced effects were significantly inhibited by IFN-γ-induced Smad7 expression. When GCTM-1 was cotransfected with AS oligonucleotide of Smad2 and Smad3, the TGF-β-induced invasion of GCTM-1 disappeared. In addition, the inhibitory effect of IFN-γ on TGF-β-dependent GCTM-1 invasion vanished by the AS oligonucleotide of Smad7 transfection. These results indicate that IFN-γ inhibits TGF-β-dependent GCTM-1 invasion through cross-talk in the Smad pathway. IFN-γ may be a new therapeutic tool for TGF-β-expressed invasive carcinomas.