Hiroki Sasaki

FGFR2 gene amplification and clinicopathological features in gastric cancer

Background:Frequency of FGFR2 amplification, its clinicopathological features, and the results of high-throughput screening assays in a large cohort of gastric clinical samples remain largely unclear.Methods:Drug sensitivity to a fibroblast growth factor receptor (FGFR) inhibitor was evaluated in vitro. The gene amplification of the FGFRs in formalin-fixed, paraffin-embedded (FFPE) gastric cancer tissues was determined by a real-time PCR-based copy number assay and fluorescence in situ hybridisation (FISH).Results:FGFR2 amplification confers hypersensitivity to FGFR inhibitor in gastric cancer cell lines. The copy number assay revealed that 4.1% (11 out of 267) of the gastric cancers harboured FGFR2 amplification. No amplification of the three other family members (FGFR1, 3 and 4) was detected. A FISH analysis was performed on 7 cases among 11 FGFR2-amplified cases and showed that 6 of these 7 cases were highly amplified, while the remaining 1 had a relatively low grade of amplification. Although the difference was not significant, patients with FGFR2 amplification tended to exhibit a shorter overall survival period.Conclusion:FGFR2 amplification was observed in 4.1% of gastric cancers and our established PCR-based copy number assay could be a powerful tool for detecting FGFR2 amplification using FFPE samples. Our results strongly encourage the development of FGFR-targeted therapy for gastric cancers with FGFR2 amplification.

MicroRNA‐148a is downregulated in gastric cancer, targets MMP7, and indicates tumor invasiveness and poor prognosis

Gastric cancer (GC) develops through deregulation of gene expression and accumulation of epigenetic abnormalities, leading to tumor cell acquisition of malignant features. MicroRNAs (miRNAs) play a critical role in cancer development where they can act as oncogenes or oncosuppressors. To identify miRNAs that are associated with some clinicopathologic features of GC and/or participate in tumor progression, miRNA expression in 20 GC tissues and five corresponding non‐neoplastic gastric mucosa was examined by miRNA microarray. Oligonucleotide array analysis was carried out for miRNA target prediction. The functions of candidate miRNAs and their target genes were also analyzed by quantitative RT‐PCR, Western blotting, reporter gene assay, and cell invasion assay. Comparison of miRNA expression profiles revealed that downregulation of miR‐148a was identified in most of the GC tissues. Downregulation of miR‐148a was significantly correlated with an advanced clinical stage, lymph node metastasis, and poor clinical outcome. Custom oligonucleotide array analysis revealed that MMP7 expression was markedly downregulated in miR‐148a‐overexpressing GC cells; MMP7 was found to be a direct and functional target of miR‐148a, participating in cell invasion. These results suggest that miR‐148a contributes to the maintenance of homeostasis in normal stomach tissue and plays an important role in GC invasion by regulating MMP7 expression.

Mesenchymal Stem Cells Provide an Advantageous Tumor Microenvironment for the Restoration of Cancer Stem Cells

Objective: Accumulating evidences suggest that cancer-associated fibroblasts are provided from bone-marrow-derived mesenchymal stem cells (BM-MSCs); however, little is known about the mechanism(s) by which BM-MSCs accelerate cancer aggressiveness. Methods: Gastric carcinoma (GC)-derived MKN-7 cells were cocultured with UE6E7T-12 BM-MSCs. The gene expression profile in MKN-7 cells was investigated by microarray analysis. Between two major types of GCs (intestinal- and diffuse-type), the expression of genes was detected by immunohistochemistry. Results: We found that direct attachment to UE6E7T-12 induced proliferation and cluster formation of MKN-7 cells. Coculture with UE6E7T-12 increased the population of CD133+ MKN-7 cells in vitro and coimplantation of these in mice resulted in subcutaneous tumors in vivo. The wingless-type MMTV integration site (WNT) family member 5A (WNT5A) and transforming growth factor-β (TGF-β)-induced (TGFBI) genes were found to be upregulated in MKN-7 cells directly attached to UE6E7T-12. Recruitment of CD271+ BM-MSC was detected preferentially in the stroma of the diffuse-type GC and this type of GC cell also showed frequent expression of WNT5A, TGF-β type I receptor and CD133. Conclusion: BM-MSC-mediated activations of the WNT and TGF-β signaling pathways were thought to provide advantageous microenvironments for cancer progression by supporting the reacquisition and maintenance of cancer stem cells.

Human Subperitoneal Fibroblast and Cancer Cell Interaction Creates Microenvironment That Enhances Tumor Progression and Metastasis

Backgrounds Peritoneal invasion in colon cancer is an important prognostic factor. Peritoneal invasion can be objectively identified as periotoneal elastic laminal invasion (ELI) by using elastica stain, and the cancer microenvironment formed by the peritoneal invasion (CMPI) can also be observed. Cases with ELI more frequently show distant metastasis and recurrence. Therefore, CMPI may represent a particular milieu that facilitates tumor progression. Pathological and biological investigations into CMPI may shed light on this possibly distinctive cancer microenvironment. Methods We analyzed area-specific tissue microarrays to determine the pathological features of CMPI, and propagated subperitoneal fibroblasts (SPFs) and submucosal fibroblasts (SMFs) from human colonic tissue. Biological characteristics and results of gene expression profile analyses were compared to better understand the peritoneal invasion of colon cancer and how this may form a special microenvironment through the interaction with SPFs. Mouse xenograft tumors, derived by co-injection of cancer cells with either SPFs or SMFs, were established to evaluate their active role on tumor progression and metastasis. Results We found that fibrosis with alpha smooth muscle actin (α-SMA) expression was a significant pathological feature of CMPI. The differences in proliferation and gene expression profile analyses suggested SPFs and SMFs were distinct populations, and that SPFs were characterized by a higher expressions of extracellular matrix (ECM)-associated genes. Furthermore, compared with SMFs, SPFs showed more variable alteration in gene expressions after cancer-cell-conditioned medium stimulation. Gene ontology analysis revealed that SPFs-specific upregulated genes were enriched by actin-binding or contractile-associated genes including α-SMA encoding ACTA2. Mouse xenograft tumors derived by co-injection of cancer cells with SPFs showed enhancement of tumor growth, metastasis, and capacity for tumor formation compared to those derived from co-injection with cancer cells and SMFs. Conclusions CMPI is a special microenvironment, and interaction of SPFs and cancer cells within CMPI promote tumor growth and metastasis.

Liver-intestine cadherin induction by epidermal growth factor receptor is associated with intestinal differentiation of gastric cancer.

Gastric cancer (GC) is one of the most common malignancies worldwide. The epidermal growth factor receptor (EGFR) molecule is very important in GC progression. To examine the correlation between EGFR and GC‐related genes, we analyzed gene expression profiles of HT‐29 cells treated with EGFR ligands and identified six genes upregulated by epidermal growth factor (EGF) and transforming growth factor (TGF)‐α treatment. Among these, we focused on cadherin 17 (CDH17) encoding liver–intestine cadherin (LI‐cadherin). Expression of LI‐cadherin was induced by both EGF and TGF‐α, as detected by quantitative RT‐PCR and Western blot analysis. A luciferase assay showed that LI‐cadherin promoter activity was enhanced by EGF or TGF‐α in both HT‐29 cells and MKN‐74 GC cells. Immunohistochemical analysis of 152 GC cases showed that out of 58 LI‐cadherin‐positive cases, 24 (41%) cases were also positive for EGFR, whereas out of 94 LI‐cadherin‐negative cases, only 9 (10%) cases were positive for EGFR (P < 0.0001). Double‐immunofluorescence staining revealed that EGFR and LI‐cadherin were coexpressed. Significant correlation was found between LI‐cadherin expression and advanced T grade and N grade. Both EGFR and LI‐cadherin expression were more frequently found in GC cases with an intestinal mucin phenotype than in cases with a gastric mucin phenotype. These results indicate that, in addition to the known intestinal transcription factor caudal type homeobox 2, EGFR activation induces LI‐cadherin expression and participates in intestinal differentiation of GC.

Gene expression signatures for identifying diffuse-type gastric cancer associated with epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT) is associated with tumor malignancy. The hedgehog-EMT pathway is preferentially activated in diffuse-type gastric cancer (GC) compared with intestinal-type GC; however, histological typing is currently the only method for distinguishing these two major types of GC. We compared the gene expression profiles of 12 bone marrow-derived mesenchymal stem cell cultures and 5 diffuse-type GC tissue samples. Numerous upregulated or downregulated genes were identified in diffuse-type GC, including CDH1, CDH2, VIM, WNT4 and WNT5. Among these genes, the mRNA ratio of CDH2 to CDH1 could distinguish the 15 diffuse-type GC samples from the 17 intestinal-type GC samples. Our results suggested that the mesenchymal features were more prominent in diffuse-type GC than in intestinal-type GC, but were weaker in diffuse-type GC than in mesenchymal stem cells. Diffuse-type GC that has undergone extensive EMT, which has a poor prognosis, can be identified by quantitative PCR analysis of only two genes.

Intraperitoneal delivery of a small interfering RNA targeting NEDD1 prolongs the survival of scirrhous gastric cancer model mice

The prognosis of patients with advanced diffuse‐type gastric cancer (GC), especially scirrhous gastric cancer (SGC) remains extremely poor. Peritoneal carcinomatosis is a frequent form of metastasis of SGC. With survival rates of patients with peritoneal metastasis at 3 and 5 years being only 9.8% and 0%, respectively, development of a new treatment is urgently crucial. For such development, the establishment of a therapeutic mouse model is required. Among the 11 GC cell lines we examined, HSC‐60 showed the most well‐preserved expression profiles of the Hedgehog and epithelial‐mesenchymal transition pathways found in primary SGCs. After six cycles of harvest of ascitic tumor cells and their orthotopic inoculation in scid mice, a highly metastatic subclone of HSC‐60, 60As6 was obtained, by means of which we successfully developed peritoneal metastasis model mice. The mice treated with small interfering (si) RNA targeting NEDD1, which encodes a gamma‐tubulin ring complex‐binding protein, by the atelocollagen‐mediated delivery system showed a significantly prolonged survival. Our mouse model could thus be useful for the development of a new therapeutic modality. Intraperitoneal administration of siRNAs of targeted genes such as NEDD1 could provide a new opportunity in the treatment of the peritoneal metastasis of SGC.

Changes in the melanocortin receptors in the hypothalamus of a rat model of cancer cachexia.

MASAMI SUZUKI, MINORU NARITA, MAHO ASHIKAWA, SADAYOSHI FURUTA, MOTOHIRO MATOBA, HIROKI SASAKI, KAZUYOSHI YANAGIHARA, KIYOSHI TERAWAKI, TSUTOMU SUZUKI, AND YASUHITO UEZONO* Division of Cancer Pathophysiology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan Department of Toxicology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan Division of Palliative Medicine and Psycho-Oncology Palliative Care Team, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan Division of Genetics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan Laboratory of Molecular Cell Biology, Department of Life Sciences, Yasuda Woman’s University Faculty of Pharmacy, 13-1 Yasuhigashi 6-chome, Asaminami-ku, Hiroshima 731-0153, Japan