Takao Hinoi

Search for transmembrane protein in gastric cancer by the Escherichia coli ampicillin secretion trap: expression of DSC2 in gastric cancer with intestinal phenotype†

Gastric cancer (GC) is one of the most common malignancies worldwide. Genes expressed only in cancer tissue, and especially on the cell membrane, will be useful molecular markers for diagnosis and may also be good therapeutic targets. To identify genes that encode transmembrane proteins present in GC, we generated Escherichia coli ampicillin secretion trap (CAST) libraries from two GC cell lines and normal stomach. By sequencing 4320 colonies from CAST libraries, we identified 30 candidate genes that encode transmembrane proteins present in GC. Quantitative reverse transcription‐polymerase chain reaction analysis of these candidates revealed that ZDHHC14, BST2, DRAM2, and DSC2 were expressed much more highly in GC than in 14 kinds of normal tissues. Among these, DSC2 encodes desmocollin 2, which is one of three known desmocollins. Immunohistochemical analysis demonstrated that 22 (28%) of 80 GC cases were positive for desmocollin 2, and desmocollin 2 expression was observed frequently in GC with the intestinal mucin phenotype. Furthermore, desmocollin 2 expression was correlated with CDX2 expression. These results suggest that expression of desmocollin 2, induced by CDX2, may be a key regulator for GC with the intestinal mucin phenotype. Our results provide a list of genes that have high potential as a diagnostic and therapeutic target for GC. Copyright

CDX2 Regulates Multidrug Resistance 1 Gene Expression in Malignant Intestinal Epithelium

The caudal-related homeobox transcription factor CDX2 has a key role in intestinal development and differentiation. CDX2 heterozygous mutant mice develop colonic polyps, and loss of CDX2 expression is seen in a subset of colon carcinomas in humans. Ectopic CDX2 expression in the stomach of transgenic mice promotes intestinal metaplasia, and CDX2 expression is frequently detected in intestinal metaplasia in the stomach and esophagus. We sought to define CDX2-regulated genes to enhance knowledge of CDX2 function. HT-29 colorectal cancer cells have minimal endogenous CDX2 expression, and HT-29 cells with ectopic CDX2 expression were generated. Microarray-based gene expression studies revealed that the Multidrug Resistance 1 (MDR1/P-glycoprotein/ABCB1) gene was activated by CDX2. Evidence that the MDR1 gene was a direct transcriptional target of CDX2 was obtained, including analyses with MDR1 reporter gene constructs and chromatin immunoprecipitation assays. RNA interference-mediated inhibition of CDX2 decreased endogenous MDR1 expression. In various colorectal cancer cell lines and human tissues, endogenous MDR1 expression was well correlated to CDX2 expression. Overexpression of CDX2 in HT-29 cells revealed increased resistance to the known substrate of MDR1, vincristine and paclitaxel, which was reversed by an MDR1 inhibitor, verapamil. These data indicate that CDX2 directly regulates MDR1 gene expression through binding to elements in the promoter region. Thus, CDX2 is probably important for basal expression of MDR1, regulating drug excretion and absorption in the lower gastrointestinal tract, as well as for multidrug resistance to chemotherapy reagent in CDX2-positive gastrointestinal cancers.

Immunohistochemical analysis of colorectal cancer with gastric phenotype: Claudin-18 is associated with poor prognosis

Claudin‐18 plays a key role in constructing tight junctions, and altered claudin‐18 expression has been documented in various human malignancies; however, little is known about the biological significance of claudin‐18 in colorectal cancer (CRC). The aim of this study is to investigate the significance of claudin‐18 expression in CRC and its association with clinicopathological factors. We performed clinicopathological analysis of claudin‐18 expression in a total of 569 CRCs by immunohistochemistry. Moreover, we investigated the association between claudin‐18 and various markers including gastric/intestinal phenotype (MUC5AC, MUC6, MUC2 and CD10), CDX2, claudin‐3, claudin‐4, p53 and Ki‐67.

Primary lung cancer presenting with metastasis to the colon: a case report

Although about 50% of lung cancers have distant metastasis at the time of initial diagnosis, colonic metastases are extremely rare. This report presents a rare clinical case of colonic metastasis from primary squamous cell carcinoma of the lung.A 60-year-old female with anorexia and fatigue was referred to the department of pulmonary surgery in our hospital. The patient was diagnosed with primary squamous cell carcinoma of the lung, T2b N3 M1b Stage IV, and chemoradiotherapy was initiated. This treatment led to a good partial response in the primary lung lesion without any new metastatic lesions.The patient developed left abdominal pain due to a bulky sigmoid colon tumor 6u2009months later, and was preoperatively diagnosed with primary colon cancer. She underwent colonic resection, and the pathology specimen demonstrated poorly differentiated squamous cell carcinoma that was suspected to be colonic metastasis from the primary lung cancer. The postoperative course was uneventful, and she was discharged. Chemotherapy for the lung cancer was scheduled in the department of pulmonary surgery.This report presented a rare case of colonic metastasis from lung cancer. When patients with advanced primary lung cancer complain of abdominal symptoms, we should consider gastrointestinal tract metastasis from lung cancer.

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.

Preservation of peritoneal fibrinolysis owing to decreased transcription of plasminogen activator inhibitor-1 in peritoneal mesothelial cells suppresses postoperative adhesion formation in laparoscopic surgery

BACKGROUNDnPostoperative adhesion formation is regulated by peritoneal fibrinolysis, which is determined by tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). This study compared peritoneal fibrinolysis and adhesion formation after laparoscopic surgery (LAP) and open surgery (OP).nnnMETHODSnWe divided 154 male rats into 3 groups after cecal cauterization: Control, no treatment; LAP, CO2 pneumoperitoneum at 5 mmHg for 60 minutes; and OP, laparotomy for 60 minutes. Adhesions were quantified at day 7. The activity and mRNA level of tPA and PAI-1 were determined by enzyme-linked immunosorbent assay in plasma and peritoneal lavage and by real-time polymerase chain reaction in peritoneal mesothelial cells from omentum. We also examined peritoneal fibrinolysis in human gastric cancer patients treated with LAP (n = 14) or OP (n = 10).nnnRESULTSnIn the animal study, adhesion scores, PAI-1 activity in peritoneal lavage fluid, and PAI-1 mRNA levels in peritoneal mesothelium were significantly greater in the OP group than the control and LAP groups. In the human study, postoperative PAI-1 mRNA levels were significantly greater in the OP group than the LAP group. Additionally, PAI-1 mRNA levels and subsequent adhesion formation were induced by prolonged operative time in the OP group, but not the LAP group.nnnCONCLUSIONnPreservation of peritoneal fibrinolysis owing to decreased PAI-1 expression at the transcriptional level in peritoneal mesothelial cells is associated with suppression of postoperative adhesion formation in LAP. PAI-1 mRNA levels and subsequent adhesion formation were not induced by prolonged operative time in LAP. These results highlight the less invasiveness nature of LAP.

Reg IV Is a Direct Target of Intestinal Transcriptional Factor CDX2 in Gastric Cancer

REG4, which encodes Reg IV protein, is a member of the calcium-dependent lectin superfamily and potent activator of the epidermal growth factor receptor/Akt/activator protein-1 signaling pathway. Several human cancers overexpress Reg IV, and Reg IV expression is associated with intestinal phenotype differentiation. However, regulation of REG4 transcription remains unclear. In the present study, we investigated whether CDX2 regulates Reg IV expression in gastric cancer (GC) cells. Expression of Reg IV and CDX2 was analyzed by Western blot and quantitative reverse transcription–polymerase chain reaction in 9 GC cell lines and 2 colon cancer cell lines. The function of the 5′-flanking region of the REG4 gene was characterized by luciferase assay. In 9 GC cell lines, endogenous Reg IV and CDX2 expression were well correlated. Using an estrogen receptor-regulated form of CDX2, rapid induction of Reg IV expression was observed in HT-29 cells. Reporter gene assays revealed an important role in transcription for consensus CDX2 DNA binding elements in the 5′-flanking region of the REG4 gene. Chromatin immunoprecipitation assays showed that CDX2 binds directly to the 5′-flanking region of REG4. These results indicate that CDX2 protein directly regulates Reg IV expression.

Adequate lymph node examination is essential to ensure the prognostic value of the lymph node ratio in patients with stage III colorectal cancer

PurposeThis study aimed to assess the prognostic value of the lymph node ratio (LNR), estimated by dividing the number of positive lymph nodes (LNs) by the number of LNs examined, for stage III colorectal cancer in comparison to the new tumor, nodes, and metastasis (TNM) system, and to evaluate the relationship between the number of LNs examined and the prognostic value of the LNR.MethodsWe retrospectively reviewed the clinicopathological data of a cohort of 266 patients with stage III colorectal cancer. We assessed the impact of LNR on the prediction of cancer recurrence in comparison to the TNM system, as well as the prognostic value of LNR in patients with a low LN count.ResultsIn multivariate analysis, the LNR was found to be an independent risk factor of cancer recurrence. The application of the LNR, in addition to the new TNM system, was more predictive of survival than the TNM system alone. A prognostic separation by LNR was observed in patients who had an adequate number of LNs examined, but not in patients with a low LN count.ConclusionsA stronger prognostic separation can be obtained by using the LNR together with the new TNM system. Adequate lymph node examination is important to ensure the prognostic value of LNR in patients with stage III colorectal cancer.

Malignant peripheral nerve sheath tumor arising from the greater omentum: Case report

Malignant peripheral nerve sheath tumors (MPNSTs) are rare soft tissue tumors that arise from a peripheral nerve or exhibit nerve sheath differentiation. Most of these tumors arise on the trunk, extremities, or head and neck regions; they are very rarely located in the abdominal cavity. The patient was a 71-year-old man who was referred to our hospital for a mass and pain in the right lower abdomen. Abdominal computed tomography revealed a large (9 × 9 cm), well-circumscribed, lobulated, heterogeneously enhanced mass in the pelvis. Exploratory laparotomy revealed a large mass in the greater omentum, and the tumor was completely excised. Histopathological analysis revealed that the tumor was composed of spindle cells with high mitotic activity. On staining the tumor, positive results were obtained for S-100 but negative results were obtained for c-kit, cluster of differentiation (CD)34, α-smooth muscle actin, and desmin. These findings strongly supported a diagnosis of MPNST primarily arising from the greater omentum. To the best of our knowledge, this is the first reported case of an MPNST arising from the greater omentum. In this report, we have described the case of a patient with an MPNST arising from the greater omentum and have discussed the clinical characteristics and management of MPNSTs.

External Validation of Two Nomograms for Predicting Patient Survival After Hepatic Resection for Metastatic Colorectal Cancer

BackgroundTwo nomograms are available for predicting patient survival after hepatic resection for metastatic colorectal cancer (CRC). However, they have not been externally validated using other databases, and so their universal applicability has not been established. We aimed to examine the validity of these nomograms for predicting patient survival after hepatic resection for metastatic CRC in different institutions.MethodsWe analyzed the cases of 113 patients who underwent hepatic resection for metastatic CRC at Hiroshima University Hospital between 1995 and 2006. In this patient set, we assessed the predictive value of the Kattan nomogram of the Memorial Sloan-Kettering Cancer Center (MSKCC) (United States) and the Kanemitsu nomogram from the Aichi Cancer Center (Japan). The concordance index was used as an accuracy measure for comparing these two nomograms. The predictive accuracy of these nomograms was compared with that of conventional predictive models.ResultsThe 3-, 5-, and 10-year overall survival rates in our cohort were 66.3%, 52.4%, and 42.7%, respectively. The concordance indexes of the pre- and postoperative Kanemitsu nomogram and that of the Kattan nomogram were 0.70, 0.69, and 0.68, respectively. These values were higher than those obtained using other models for hepatic metastatic CRC, including the clinical risk score of the MSKCC and the grading system of the Japanese Society for Cancer of the Colon and Rectum.ConclusionsThe high predictive accuracy of both nomograms shows that these predictive tools can be used in different institutions. Patient counseling and adjuvant therapy decision-making should benefit from use of these nomograms.