Michio Kaminishi

Relation between microRNA expression and progression and prognosis of gastric cancer: a microRNA expression analysis

BACKGROUND Analyses of microRNA expression profiles have shown that many microRNAs are expressed aberrantly and correlate with tumorigenesis, progression, and prognosis of various haematological and solid tumours. We aimed to assess the relation between microRNA expression and progression and prognosis of gastric cancer. METHODS 353 gastric samples from two independent subsets of patients from Japan were analysed by microRNA microarray. MicroRNA expression patterns were compared between non-tumour mucosa and cancer samples, graded by diffuse and intestinal histological types and by progression-related factors (eg, depth of invasion, metastasis, and stage). Disease outcome was calculated by multivariable regression analysis to establish whether microRNAs are independent prognostic factors. FINDINGS In 160 paired samples of non-tumour mucosa and cancer, 22 microRNAs were upregulated and 13 were downregulated in gastric cancer; 292 (83%) samples were distinguished correctly by this signature. The two histological subtypes of gastric cancer showed different microRNA signatures: eight microRNAs were upregulated in diffuse-type and four in intestinal-type cancer. In the progression-related signature, miR-125b, miR-199a, and miR-100 were the most important microRNAs involved. Low expression of let-7g (hazard ratio 2.6 [95% CI 1.3-4.9]) and miR-433 (2.1 [1.1-3.9]) and high expression of miR-214 (2.4 [1.2-4.5]) were associated with unfavourable outcome in overall survival independent of clinical covariates, including depth of invasion, lymph-node metastasis, and stage. INTERPRETATION MicroRNAs are expressed differentially in gastric cancers, and histological subtypes are characterised by specific microRNA signatures. Unique microRNAs are associated with progression and prognosis of gastric cancer. FUNDING National Cancer Institute.

Improvement of cancer-targeting therapy, using nanocarriers for intractable solid tumors by inhibition of TGF-β signaling

Transforming growth factor (TGF)-β plays a pivotal role in regulation of progression of cancer through effects on tumor microenvironment as well as on cancer cells. TGF-β inhibitors have recently been shown to prevent the growth and metastasis of certain cancers. However, there may be adverse effects caused by TGF-β signaling inhibition, including the induction of cancers by the repression of TGF-β-mediated growth inhibition. Here, we present an application of a short-acting, small-molecule TGF-β type I receptor (TβR-I) inhibitor at a low dose in treating several experimental intractable solid tumors, including pancreatic adenocarcinoma and diffuse-type gastric cancer, characterized by hypovascularity and thick fibrosis in tumor microenvironments. Low-dose TβR-I inhibitor altered neither TGF-β signaling in cancer cells nor the amount of fibrotic components. However, it decreased pericyte coverage of the endothelium without reducing endothelial area specifically in tumor neovasculature and promoted accumulation of macromolecules, including anticancer nanocarriers, in the tumors. Compared with the absence of TβR-I inhibitor, anticancer nanocarriers exhibited potent growth-inhibitory effects on these cancers in the presence of TβR-I inhibitor. The use of TβR-I inhibitor combined with nanocarriers may thus be of significant clinical and practical importance in treating intractable solid cancers.

Gastric cancer treated in 1991 in Japan: data analysis of nationwide registry

The Japanese Gastric Cancer Association Registration Committee reported the treatment results and causes of death of patients with primary gastric cancer treated in 1991 at the leading hospitals in Japan. Data of 8851 patients with primary gastric cancer were collected from 113 hospitals, and data of 7935 patients with gastric resection were finally analyzed. The lost-to-follow-up rate was 6.9%; the direct death rate was 1.0%. The cumulative 5-year survival rate (5YSR) of all the patients was 68.2%; 89.9% for Stage I, 69.1% for Stage II, 43.5% for Stage III, and 9.9% for Stage IV. Characteristic findings of the analyzed data were (1) high proportion of early-stage cancer, (2) high resection rate, (3) low mortality rate, (4) low incidence of upper-third cancer, (5) poor treatment results in cases with scirrhous cancer, infiltrating growth, and marked lymphatic or venous invasion, and (6) predominance of systematic (D2) and extended lymphadenectomies possibly resulting in reducing local recurrence and improving survivals.

Lysyl oxidase is a tumor suppressor gene inactivated by methylation and loss of heterozygosity in human gastric cancers.

Lysyl oxidase (LOX) and HRAS-like suppressor (HRASLS) are silenced in human gastric cancers and are reported to have growth-suppressive activities in ras-transformed mouse/rat fibroblasts. Here, we analyzed whether or not LOX and HRASLS are tumor suppressor genes in human gastric cancers. Loss of heterozygosity and promoter methylation of LOX were detected in 33% (9 of 27) and 27% (26 of 96) of gastric cancers, respectively. Biallelic methylation and loss of heterozygosity with promoter methylation were also demonstrated in gastric cancers. Silencing of LOX was also observed in colon, lung, and ovarian cancer cell lines. As for mutations, only one possible somatic mutation was found by analysis of 96 gastric cancer samples and 58 gastric and other cancer cell lines. When LOX was introduced into a gastric cancer cell line, MKN28, in which LOX and HRASLS were silenced, it reduced the number of anchorage-dependent colonies to 57 to 61%, and the number of anchorage-independent colonies to 11 to 23%. Sizes of tumors formed in nude mice were reduced to 19 to 26%. Growth suppression in soft agar assay was also observed in another gastric cancer cell line, KATOIII. On the other hand, neither loss of heterozygosity nor a somatic mutation was detected in HRASLS, and its introduction into MKN28 did not suppress the growth in vitro or in vivo. These data showed that LOX is a tumor suppressor gene inactivated by methylation and loss of heterozygosity in gastric cancers, and possibly also in other cancers.

Effect of early eradication on Helicobacter pylori-related gastric carcinogenesis in Mongolian gerbils

Helicobacter pylori (Hp) infection and gastric carcinogenesis are known to have a close relationship, but the effect of eradication of Hp on Hp‐related gastric carcinogenesis has not been fully studied experimentally. To evaluate the effect of eradication in gastric carcinogenesis, an experimental model with eradication in the early, middle or late period was studied using Hp‐infected and N‐methyl‐N‐nitrosourea (MNU)‐treated Mongolian gerbils. The animals were divided into seven groups: group A (MNU+Hp+ eradication at 15 w), group B (MNU+Hp+eradication at 35 w), group C (MNU+Hp+eradication at 55 w), group D (MNU+Hp), group E (MNU), group F (Hp) and group G (control). The tumor incidences at week 75 in the early (group A), middle (group B) and late (group C) eradicated groups were 6.7%, 27.3% and 38.2%, respectively. The incidence of 56.3% in the non‐eradicated group was the highest (group D). Incidences in group E, group F and group G were 6.3%, 0.0% and 0.0%, respectively. The tumor incidences were related to the period of inflammatory status induced by Hp infection. Hp infection strongly enhanced gastric carcinogenesis initiated with a chemical carcinogen, and following eradication at an early period this enhancing effect was effectively reduced. Eradication at an early stage of inflammation might be effective in preventing Hp‐related gastric carcinogenesis. (Cancer Sci 2003; 94: 235–239)

Inhibition of Cyclooxygenase-2 Suppresses Lymph Node Metastasis via Reduction of Lymphangiogenesis

Cyclooxygenase-2 (COX-2) inhibitor has been reported to suppress tumor progression. However, it is unclear whether this inhibitor can also prevent lymphatic metastasis. To determine the effects of COX-2 inhibitor on lymphatic metastasis, etodolac, a COX-2 inhibitor, was given p.o. to mice bearing orthotopic xenografts or with carcinomatous peritonitis induced with a highly metastatic human diffuse-type gastric carcinoma cell line, OCUM-2MLN. Tumor lymphangiogenesis was significantly decreased in etodolac-treated mice compared with control mice. Consistent with this decrease in lymphangiogenesis, the total weight of metastatic lymph nodes was less in etodolac-treated mice than in control mice. Immunohistochemical analysis revealed that the major source of vascular endothelial growth factor-C (VEGF-C) and VEGF-D was F4/80-positive macrophages in our models. The mRNA levels of VEGF-C in mouse macrophage-like RAW264.7 cells, as well as those in tumor tissues, were suppressed by etodolac. The growth of human dermal lymphatic microvascular endothelial cells was also suppressed by etodolac. Supporting these findings, etodolac also inhibited lymphangiogenesis in a model of chronic aseptic peritonitis, suggesting that COX-2 can enhance lymphangiogenesis in the absence of cancer cells. Our findings suggest that COX-2 inhibitor may be useful for prophylaxis of lymph node metastasis by reducing macrophage-mediated tumor lymphangiogenesis.

Manipulation of the small intestine as a cause of the increased inflammatory response after open compared with laparoscopic surgery

Laparoscopic surgery of the gastrointestinal tract involves a reduced immune response compared with open surgery. The aim of this study was to assess manual handling of the gut in open procedures as the principal cause of the enhanced immune response.

Frequent hypomethylation in multiple promoter CpG islands is associated with global hypomethylation, but not with frequent promoter hypermethylation

Hypomethylation of the global genome, considered to be composed mainly of repetitive sequences, is consistently observed in cancers, and aberrant hypo‐ and hypermethylation of CpG islands (CGIs) in promoter regions are also observed. Since methylation alterations in unique promoter sequences and in other genomic regions have distinct consequences, we analyzed the relationship between the global hypomethylation and the hypomethylation of unique promoter CGIs using human gastric cancers. Seven of ten gastric cancer cell lines showed marked decreases in 5‐methylcytosine content, which correlated with hypomethylation of the LINE1 repetitive sequence. Six of the seven cell lines showed hypomethylation in five or all of the six normally methylated CGIs in promoter regions of six genes, and this was associated with induction of aberrant expression. The remaining three cell lines without global hypomethylation showed promoter hypomethylation in one or none of the six CGIs. Frequent promoter hypomethylation, however, did not correlate with frequent promoter hypermethylation. In primary gastric cancers too, global hypomethylation was associated with hypomethylation of LINE1 repetitive sequence and promoter hypomethylation. Of 93 gastric cancers, 33 cancers with frequent promoter hypomethylation and 27 cancers with frequent promoter hypermethylation constituted different groups. These findings represent experimental evidence that frequent hypomethylation of normally methylated promoter CGIs is associated with global hypomethylation, and that these hypomethylations occur independently of frequent promoter CGI hypermethylation. (Cancer Sci 2004; 95: 58–64)

The presence of aberrant DNA methylation in noncancerous esophageal mucosae in association with smoking history: a target for risk diagnosis and prevention of esophageal cancers.

Esophageal squamous cell carcinomas (ESCCs) tend to have multiple primary lesions, and it is believed that they arise from background mucosae with accumulation of genetic/epigenetic alterations. In this study, the objective was to elucidate the effects of smoking and drinking on the accumulation of epigenetic alterations in background mucosae.

Induction of Glandular Stomach Cancers in Helicobacter pylori-sensitive Mongolian Gerbils Treated with N-Methyl-N-nitrosourea and N-Methyl-N′-nitro-N-nitrosoguanidine in Drinking Water

An animal model of stomach carcinogenesis was established using Mongolian gerbils with N‐methyl‐N‐nitrosourea (MNU) and N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG) as the carcinogens. In addition, the sensitivity of these gerbils to Helicobacter pylori (H. pylori) was confirmed. One hundred and sixty specific pathogen‐free male MGS/Sea animals, 7 weeks old, were treated with MNU in the drinking water (30 ppm for alternate weeks to give 10 weeks exposure, or 10 ppm or 3 ppm for 20 weeks continuous exposure), or given MNNG in the drinking water at 400 ppm or 200 ppm for 20 weeks, or orally inoculated with ATCC43504 H. pylori (1.7×1088 CFUs/animal). Adenocarcinomas in the glandular stomach were found in 2 out of 12 effective animals (2/12) treated with 30 ppm MNU at week 20, although all were dead or moribund by week 30 due to MNU toxicity. At week 50, the incidences of gastric adenocarcinomas in groups treated with 10 ppm MNU, 3 ppm MNU, 400 ppm MNNG, and 200 ppm MNNG were 2/21 (9.5%), 1/23 (4.3%), 7/11 (63.6%), and 1/10 (10.0%). The lesions were generally well differentiated, although poorly differentiated adenocarcinoma was also found in a single gerbil in each of the 10 ppm MNU and 400 ppm MNNG groups. In control animals no tumors were found. In the infection study, the animals were killed at week 20, and H. pylori was detected in all cases, causing multiple erosions with marked inflammatory cell infiltration in the lamina propria and submucosa, and frequent formation of lymphoid follicles. Thus, MNU and MNNG in the drinking water induced neoplastic lesions in the glandular stomach epithelium of H. pylori‐sensitive gerbils.