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The receptor for advanced glycation end-products (RAGE) is a newly recognized factor regulating cancer cell invasion and metastasis. This study investigated the expression of RAGE in gastric carcinomas and its association with invasion and metastasis. Of eight gastric cancer cell lines examined, seven constitutively expressed RAGE messenger ribonucleic acid (mRNA), MKN45 being the exception. RAGE protein expression of MKN28 cells treated with RAGE antisense S-oligodeoxynucleotide was nine times less than that of sense S-oligodeoxynucleotide-treated cells. Growth of cells under RAGE antisense S-oligodeoxynucleotide treatment was not different from that seen under sense S-oligodeoxynucleotide treatment in MKN28 (a cell line producing high levels of RAGE) and MKN45 (a non-RAGE-expressing cell line). RAGE antisense S-oligodeoxynucleotide treatment suppressed the invasive activity of RAGE-positive MKN28 cells, as estimated by in vitro invasion assay. The number of MKN28 cells invading the type IV collagen-coated membrane under RAGE antisense S-oligodeoxynucleotide treatment was significantly lower than under RAGE sense S-oligodeoxynucleotide treatment (p<0.0001). In contrast, antisense and sense S-oligodeoxynucleotide-treated RAGE-negative MKN45 cells showed no difference. A wound-healing assay showed that no RAGE antisense S-oligodeoxynucleotide-treated MKN28 cells migrated into the scraped area, whereas sense S-oligodeoxynucleotide-treated cells showed many budding nests in the scraped area. Immunohistochemistry of gastric carcinoma tissue showed that 62 (65%) of the 96 cases examined were RAGE-positive and that poorly differentiated adenocarcinomas preferentially expressed RAGE protein (38/42, 90%) (p<0.0001). Strong RAGE immunoreactivity was also correlated with depth of invasion and lymph node metastasis (p<0.0001). RAGE-positive cancer cells tended to be distributed at the invasive front of primary tumours and were detected in all metastatic foci in lymph nodes. In contrast, a major RAGE ligand, amphoterin, was expressed in 82 (85%) of the 96 cases, regardless of histological type and disease progression. RAGE expression appears to be closely associated with invasion and metastasis in gastric cancer.

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(HY) S U M M A R Y Among tight-junction proteins, claudins, which play a key role in paracellular transport across epithelia, claudins 1 to 5 are expressed in the intestine, and changes in their abundance and/or distribution are considered to contribute to various gastrointestinal diseases. We investigated, by reverse transcription-PCR, immunoblot, and immunofluorescence analyses, which other claudin species were expressed in the mouse intestine, and whether they showed unique expression profiles. Rabbit polyclonal antibodies against mouse claudin-8, claudin-12, and claudin-15 were generated, and their specificity was verified by immunoblotting using COS-7 cells transfected with individual claudin cDNAs. Claudin-7, -8, -12, -13, and -15 appeared to be expressed in the duodenum, jejunum, ileum, and/or colon with remarkable variations in the expression levels along the intestinal tract, and had distinct subcellular localization in the intestinal epithelium. In addition, claudin-13 and -15 exhibited gradients along the crypt-surface axis of the colon. By contrast,

The expressions of epidermal growth factor (EGF) and its receptor were studied immunohistochemically in a total of 156 gastric carcinomas; 26 early and 130 advanced. No EGF immunoreactivity was found in early carcinomas, while EGF-positive tumor cells were detected in 38 (29.2%) of the 130 advanced carcinomas. EGF receptor immunoreactivity was detected in one (3.8%) of the 26 early carcinomas and in 44 (33.8%) of the 130 advanced carcinomas, the incidence being significantly different (p less than 0.01). Out of the 130 advanced carcinomas, 17 (13.1%) had synchronous expression of EGF and its receptor and most of the tumors with strong expression of EGF were positive to EGF receptor. A significant correlation was observed between the depth of tumor invasion and EGF or its receptor immunoreactivity in tumor cells (p less than 0.05). Furthermore, a good correlation was demonstrated between the synchronous expression of EGF and its receptor and the depth of tumor invasion or the tumor staging. The incidence of cases with EGF in metastatic tumors was significantly higher than that in primary tumors (p less than 0.05). Patients with synchronous expression of EGF and its receptor had a far poorer prognosis than those without EGF and receptor.

Purpose: Overexpression of PRL-3 has been implicated in colorectal cancer metastases. We investigated the significance of PRL-3 expression in the progression and development of colorectal cancer.Experimental Design: We transfected PRL-3-specific small interfering RNA into human colon cancer DLD-1 cells and analyzed its effect on proliferation, motility, and hepatic colonization. Using an in situ hybridization method, we examined the levels of PRL-3 expression in both primary (177 cases) and metastatic (92 cases) human colorectal cancers and elucidated the relationships with clinicopathological parameters including the incidence of metachronous liver and/or lung metastasis after curative surgery for primary tumor.Results: Transient down-regulation of PRL-3 expression in DLD-1 cells abrogated motility (in vitro) and hepatic colonization (in vivo), but no effect on the proliferation of these cells was observed. In human primary colorectal cancers, the frequency of up-regulated PRL-3 expression in cases with liver (84.4%) or lung (88.9%) metastasis was statistically higher than that in cases without either type of metastasis (liver, 35.9%; lung, 42.3%). In metastatic colorectal cancer lesions, high expression of PRL-3 was frequently detected (liver, 91.3%; lung, 100%). Interestingly, metachronous metastasis was observed more frequently in the cases with high PRL-3 expression (P < 0.0001). Conclusions:These results indicate that PRL-3 expression in colorectal cancers may contribute to the establishment of liver metastasis, particularly at the step in which cancer cells leave the circulation to extravasate into the liver tissue. In addition, PRL-3 is expected to be a promising biomarker for identifying colorectal cancer patients at high risk for distant metastases.

Tumor associated macrophages (TAMs) are the most abundant cancer stromal cells educated by tumor microenvironment to acquire trophic functions facilitating angiogenesis, matrix breakdown and cancer cell motility. Tumor associated macrophages have anti-inflammatory properties or "alternatively" activated (M2) phenotype expressing CD204 and ⁄ or CD163. To know the role of TAMs in the growth and progression of esophageal squamous cell carcinomas (ESCCs), we calculated intratumoral CD204, CD163 or CD68 expressing macrophage count (M/C) and CD34-positive microvessel density (MVD) by immunohistochemistry in 70 cases of surgically resected ESCCs and compared them with the clinicopathological factors and prognosis of patients. M/C had positive linear association with MVD. High CD204 + M/C were significantly correlated with more malignant phenotypes including depth of tumor invasion, lymph and blood vessel invasion, lymph node metastasis as well as clinical stages. On the other hand, CD163+ M/C did not associate with these clinicopathological factors with the exception of depth of tumor invasion and blood vessel invasion. Patients with high CD204 + M/C ESCCs showed poor disease-free survival (P = 0.021). Conditioned media of five ESCC cell lines (TE-8, -9, -10, -11 and -15) induced mRNA as well as protein expression of CD204 but not of CD163 with upregulation of vascular endothelial growth factor-A mRNA in TPA treated human acute monocytic leukemia cell line THP-1. These results overall indicate that CD204 is a useful marker for TAMs contributing to the angiogenesis, progression and prognosis of ESCCs whose specific tumor microenvironment may educate macrophages to be CD204

Overexpression of the enhancer of zeste homolog 2 (EZH2) protein, a known repressor of gene transcription, has been reported to be associated with biological malignancy of prostate cancer and several other cancers. The purpose of this study was to examine the expression of EZH2 and analyze its relationship with the clinicopathological features of human gastric cancers. Expression levels of EZH2 mRNA and protein were examined in 13 gastric cancer cell lines and in 83 surgically removed human gastric cancer tissues. Immunohistochemical analysis of the 83 tissue samples and corresponding non-cancerous gastric mucosa showed that EZH2 was more highly expressed in the cancerous than in the non-cancerous tissues, and the expression levels of EZH2 were highly correlated with tumor size, depth of invasion, vessel invasion, lymph node metastasis and clinical stages. Univariate analysis of survival rate calculated by the KaplanMeier method revealed that gastric cancer patients with high-level EZH2 expression had poorer prognosis than those expressing no or low levels of EZH2 (P = 0.0271). These findings suggest that overexpression of EZH2 may contribute to the progression and oncogenesis of human gastric cancers, and thus immunohistochemical study of EZH2 expression may serve as a new biomarker for predicting the prognosis of gastric cancers. (Cancer Sci 2006; 97: 484-491) A lthough gastric cancer has gradually decreased in prevalence, it still accounts for a large portion of cancerrelated deaths in Japan. The most informative prognostic factor is the tumor stage, which involves both the depth of invasion and the extent of metastasis. The size and histological type of the tumor may also be useful factors. Despite the complexity of stomach carcinogenesis, a number of molecular studies have been performed in a search for additional prognostic factors. As a result, several proteins, such as transforming growth factor alpha (TGFα), epidermal growth factor receptor (EGFR), c-met, c-erbB2, cyclin E, p27Kip1 and CDC25B, have been identified as markers of the malignancy of gastric cancer.(1-3) The search for molecular factors that are highly correlated with prognosis may lead to the discovery of factors that can help to predict not only patient survival, but also the tumor response to specific anticancer drugs. One new marker that has potential for cancer screening and can be a predictor of patient survival is enhancer of zeste homolog 2 (EZH2). EZH2, also called histone lysine methyltransferase (HKMT), was cloned as one of the polycomb group genes. (4) The function of EZH2 is to catalyze the subunit of the polycomb repressor complex by methylating lysine 9 and 27 of histone H3.(5-8) Although EZH2, which by itself lacks enzyme activity, it is assumed to associate with specific polypeptides present in the polycomb repressive complexes 2 and 3 (PRC2/3), constructing an EZH2 complex to work as a repressor gene in various organs. (5)(6)(7)(8)(9)(10)(11)(12) Recently, close correlation between overexpression of EZH2 and progression of ...