Kazuhiro Sentani

Serum olfactomedin 4 (GW112, hGC‐1) in combination with Reg IV is a highly sensitive biomarker for gastric cancer patients

Gastric cancer (GC) is 1 of the most common human cancers. Early detection remains the most promising approach to improving long‐term survival of patients with GC. We previously performed Serial Analysis of Gene Expression (SAGE) on 4 primary GCs and identified several GC‐specific genes including Reg IV. Of these genes, olfactomedin 4 (OLFM4, also known as GW112 or hGC‐1) is a candidate gene for cancer‐specific expression. In this study, we examined the expression of olfactomedin 4 in human GC by immunohistochemistry. We also assessed serum olfactomedin 4 levels in GC patients by enzyme‐linked immunosorbent assay. 94 (56%) of 167 GC cases were positive for olfactomedin 4 by immunostaining. Olfactomedin 4 staining was observed more frequently in stage I/II cases than in stage III/IV cases. The serum olfactomedin 4 concentration in presurgical GC patients (n = 123, mean ± SE, 36.3 ± 3.5 ng/mL) was significantly higher than that in healthy individuals (n = 76, 16.6 ± 1.6 ng/mL). In patients with stage I GC, the sensitivity of serum olfactomedin 4 (25%) and Reg IV (35%) was superior to that of CA19‐9 (5%) or CEA (3%). Furthermore, in patients with stage I GC, the combination of olfactomedin 4 and Reg IV elevated the diagnostic sensitivity to 52%. These results suggest that serum olfactomedin 4 is a useful marker for GC and its measurement alone or in combination with Reg IV has utility in the early detection of GC.

Molecular pathology of gastric cancer: research and practice.

Recent advances in the understanding of molecular stomach carcinogenesis are reviewed. As to molecular events in individual mucin phenotypes of gastric cancer, the CDX2-Reg IV-SOX9 pathway is associated with the intestinal mucin phenotype, while OLFM4 and CLDN18 are novel markers for the gastric phenotype. microRNAs play an important role in epigenetic deregulation in gastric cancer. Many microRNAs are up-regulated and down-regulated, and some of these are associated with histological differentiation and cancer progression. Reduced miR-200 may participate in the genesis of diffuse type gastric cancer by reducing E-cadherin expression. Genetic polymorphism is a crucial endogenous cause and a fundamental factor of cancer risk. PSCA polymorphism alters the susceptibility to diffuse type gastric cancer through modulation of cell proliferation activity. Cancer stem cells possess the capacity for self-renewal and cause the heterogeneous lineages of cancer cells. Cancer stem cells also show resistance to anti-tumor chemotherapy. Only a minor population of gastric cancer cells reveals the properties of cancer stem cells, and CD44 is one of the markers for gastric cancer stem cells. The origin of gastric cancer stem cells remains to be elucidated.

Serum Concentration of Reg IV in Patients with Colorectal Cancer: Overexpression and High Serum Levels of Reg IV Are Associated with Liver Metastasis

Objective: Regenerating islet-derived family, member 4 (regenerating gene type IV, Reg IV) is overexpressed in colorectal cancer (CRC). The aim of this study was to investigate the diagnostic utility of Reg IV determination in sera from patients with CRC. Methods: We examined the expression and distribution of Reg IV in CRC by immunohistochemistry and determined Reg IV levels in sera from patients with CRC by enzyme-linked immunosorbent assay. Results: Immunostaining revealed that 23 of 80 (29%) CRC cases were positive for Reg IV. CRC cases with metastatic recurrence in the liver showed more frequently Reg IV staining than those without (p = 0.0102). Patients with CRC showing Reg IV staining had a significantly worse survival than those without Reg IV staining (p = 0.0117). Preoperatively, serum Reg IV concentrations were not elevated in CRC patients at stage 0–III, being in contrast to the significantly increased preoperative levels in stage IV CRC patients with liver metastasis. Conclusion: These results suggest that Reg IV is a prognosticator for poor survival. Serum Reg IV concentration may predict CRC recurrence in the liver.

Increased expression but not genetic alteration of BRG1, a component of the SWI/SNF complex, is associated with the advanced stage of human gastric carcinomas.

BRG1, a component of the SWI/SNF complex, regulates gene transcription through chromatin remodeling. Certain human cancer cell lines have been shown to contain homozygous deletions or mutations, half of which are concentrated in exons 4 and 10, resulting in aberrant BRG1 expression. We examined the expression of BRG1 in 38 gastric carcinomas and corresponding nonneoplastic mucosa by using the quantitative real-time RT-PCR method. Twenty-three carcinomas (61%) showed increased BRG1 expression in tumor tissue in comparison with that in nonneoplastic mucosa. The T/N ratio (the expression level of BRG1 mRNA in tumor tissues relative to those in corresponding nonneoplastic mucosa) in advanced cases of gastric carcinoma (stages III and IV) was significantly higher than that in cases of stage I and II carcinoma (p = 0.029). Furthermore, gastric carcinomas with lymph node metastasis showed a tendency to express BRG1 at a higher level than gastric carcinomas without metastasis (p = 0.097). We also searched for genetic alterations of the BRG1 gene in 8 gastric carcinoma cell lines and 33 primary gastric carcinomas by PCR-SSCP analysis. No SSCP variants in exons 4, 10 and 16 of the BRG1 gene were found in both gastric carcinoma cell lines and primary gastric carcinomas. These results suggest that, although genetic abnormality of BRG1 might be rare, an increased expression of BRG1 might be associated with the development and progression of gastric carcinoma.

Molecular pathobiology of gastric cancer.

Gastric carcinogenesis is a multistep process, during which numerous genetic and epigenetic alterations accumulate: there are abnormalities of growth factors/receptors, angiogenic factors, cell cycle regulators, DNA mismatch repair genes etc. These abnormalities define, at the same time, the biological character of the cancer cell and may thus serve as therapeutic targets. Genetic instability may cause accumulation of genetic abnormalities. The most important epigenetic alterations are DNA methylation, histone modification and chromatin remodeling. Some of these changes are common in gastric cancer, regardless of subtype, and some differ by histological type or (gastric or intestinal) mucin phenotype. Genetic polymorphism is a crucial endogenous cause and fundamental aspect of cancer risk. Importantly, genetic polymorphisms are also associated with the therapeutic efficacy and toxicity of anti-cancer drugs. Genomic science and technology such as Serial Analysis of Gene Expression (SAGE) allows the identification of novel genes and molecules specifically up-regulated or down-regulated in gastric cancer, e.g., RegIV and claudin-18 can be identified. Advances in our understanding of the genetic and molecular bases lead to improved diagnosis, personalised medicine and prevention of gastric cancer.

Deficiency of Claudin-18 Causes Paracellular H+ Leakage, Up-regulation of Interleukin-1β, and Atrophic Gastritis in Mice

BACKGROUND & AIMS Although defects in tight junction (TJ) epithelial paracellular barrier function are believed to be a primary cause of inflammation, the mechanisms responsible remain largely unknown. METHODS We generated knockout mice of stomach-type claudin-18, a major component of TJs in the stomach. RESULTS Cldn18(-/-) mice were afflicted with atrophic gastritis that started on postnatal day 3. This coincided with a decrease in intragastric pH due to H(+) secretion from parietal cells and concomitant up-regulation of the cytokines, interleukin-1β, cyclooxygenase-2, and KC, resulting in spasmolytic polypeptide-expressing metaplasia (SPEM). Oral administration of hydrochloric acid on postnatal day 1 induced the expression of these cytokines in Cldn18(-/-) infant stomach, but not in Cldn18(+/+) mice. A paracellular H(+) leak in Cldn18(-/-) stomach was detected by electrophysiology and H(+) titration, and freeze-fracture electron microscopy showed structural defects in the TJs, in which the tightly packed claudin-18 (stomach-type)-based TJ strands were lost, leaving a loose meshwork of strands consisting of other claudin species. CONCLUSIONS These findings provide evidence that claudin-18 normally forms a paracellular barrier against H(+) in the stomach and that its deficiency causes paracellular H(+) leak, a persistent up-regulation of proinflammatory cytokines, chronic recruitment of neutrophils, and the subsequent development of SPEM in atrophic gastritis.

Expression of RhoC Is Associated with Metastasis of Gastric Carcinomas

Objectives:RhoC, a member of the ras-related small GTPase protein family, regulates cytoskeletal structures and has the potential to transform cultured cells. It has recently been reported that RhoC contributes to the metastatic phenotype of melanoma cells. The purpose of this study was to clarify its biological relevance to gastric carcinogenesis and metastasis. Methods: We examined the expression of RhoC by quantitative RT-PCR in 51 cases of gastric carcinoma tissues from prior surgical cases (intestinal type: 24 cases, diffuse type: 27 cases) and in 8 gastric carcinoma cell lines. Results:RhoC expression levels in primary tumors were significantly higher in cases with metastasis than in those without metastasis (p = 0.0202; Mann-Whitney U test). RhoC expression levels in primary tumor and their metastatic tumors were significantly higher than their corresponding nonneoplastic mucosa (p = 0.0357, and 0.0173, respectively; Wilcoxon signed rank test). RhoC mRNA expression was confirmed in the gastric carcinoma cell lines. Conclusion: Our findings suggest that elevated expression of the RhoC gene may be involved in the metastasis of gastric carcinomas and may be a good genetic marker for the prediction of a metastatic potential.

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.

Immunohistochemical staining of Reg IV and claudin-18 is useful in the diagnosis of gastrointestinal signet ring cell carcinoma.

Signet-ring cell carcinoma (SRCC) is a unique subtype of adenocarcinoma that is characterized by abundant intracellular mucin accumulation and a crescent-shaped nucleus displaced toward one end of the cell. Identification of an SRCCs primary site is important for better planning of patient management because the treatment and prognosis differs markedly depending on the origin of the SRCC. In the present study, we analyzed the immunohistochemical characteristics of 94 cases of SRCC, including 21 cases of gastric SRCC, 16 of colorectal SRCC, 10 of breast SRCC, and 47 of pulmonary SRCC, with antibodies against Reg IV and claudin-18, which we previously identified as gastric cancer-related genes. We also tested known markers cytokeratin 7, cytokeratin 20, MUC2, MUC5AC, caudal-related homeobox gene 2 (CDX2), thyroid transcription factor-1, mammaglobin, gross cystic disease fluid protein 15, and estrogen receptor. All 21 cases of gastric SRCC and 16 cases of colorectal SRCC were positive for Reg IV, and the remaining SRCCs were negative. Eighteen of 21 (86%) gastric SRCCs and 6 of 16 (38%) colorectal SRCCs were positive for claudin-18, whereas another SRCCs were negative. In conclusion, Reg IV staining and claudin-18 staining can aid in diagnosis of gastrointestinal SRCC.

Transcriptome dissection of gastric cancer: Identification of novel diagnostic and therapeutic targets from pathology specimens

Gastric cancer is the fourth most common malignancy in the world, and mortality due to gastric cancer is second only to that from lung cancer. ‘Transcriptome dissection’ is a detailed analysis of the entire expressed transcripts from a cancer, for the purpose of understanding the precise molecular mechanism of pathogenesis. Serial analysis of gene expression (SAGE) is a suitable technique for performing transcriptome dissection. Gastric cancers of different stages and histology were analyzed on SAGE, and one of the largest gastric cancer SAGE libraries in the world was created (GEO accession number GSE 545). Through SAGE, many candidate genes have been identified as potential diagnostic and therapeutic targets for the treatment of gastric cancer. Regenerating islet‐derived family, member 4 (Reg IV) participated in 5‐fluorouracil (5‐FU) resistance and peritoneal metastasis, and its expression was associated with an intestinal phenotype of gastric cancer and with endocrine differentiation. GW112 expression correlated with advanced tumor stage. Measurement of Reg IV and GW112 levels in sera indicated a sensitivity of 57% for detection of cancer. SPC18 participated in tumor growth and invasion through transforming tumor growth factor‐α upregulation. Palate, lung, and nasal epithelium carcinoma‐associated protein (PLUNC) was a useful marker for gastric hepatoid adenocarcinoma. Expression of SOX9, HOXA10, CDH17, and loss of claudin‐18 expression were associated with an intestinal phenotype of gastric cancer. Information obtained from transcriptome dissection greatly contributes to diagnosis and treatment of gastric cancer.