Hiroshi Ida

RUNX3, A Novel Tumor Suppressor, Is Frequently Inactivated in Gastric Cancer by Protein Mislocalization

Loss of RUNX3 expression is suggested to be causally related to gastric cancer as 45% to 60% of gastric cancers do not express RUNX3 mainly due to hypermethylation of the RUNX3 promoter. Here, we examined for other defects in the properties of RUNX3 in gastric cancers that express RUNX3. Ninety-seven gastric cancer tumor specimens and 21 gastric cancer cell lines were examined by immunohistochemistry using novel anti-RUNX3 monoclonal antibodies. In normal gastric mucosa, RUNX3 was expressed most strongly in the nuclei of chief cells as well as in surface epithelial cells. In chief cells, a significant portion of the protein was also found in the cytoplasm. RUNX3 was not detectable in 43 of 97 (44%) cases of gastric cancers tested and a further 38% showed exclusive cytoplasmic localization, whereas only 18% showed nuclear localization. Evidence is presented suggesting that transforming growth factor-β is an inducer of nuclear translocation of RUNX3, and RUNX3 in the cytoplasm of cancer cells is inactive as a tumor suppressor. RUNX3 was found to be inactive in 82% of gastric cancers through either gene silencing or protein mislocalization to the cytoplasm. In addition to the deregulation of mechanisms controlling gene expression, there would also seem to be at least one other mechanism controlling nuclear translocation of RUNX3 that is impaired frequently in gastric cancer.

The RUNX3 Tumor Suppressor Upregulates Bim in Gastric Epithelial Cells Undergoing Transforming Growth Factor β-Induced Apoptosis

ABSTRACT Genes involved in the transforming growth factor β (TGF-β) signaling pathway are frequently altered in several types of cancers, and a gastric tumor suppressor RUNX3 appears to be an integral component of this pathway. We reported previously that apoptosis is notably reduced in Runx3−/− gastric epithelial cells. In the present study, we show that a proapoptotic gene Bim was transcriptionally activated by RUNX3 in the gastric cancer cell lines SNU16 and SNU719 treated with TGF-β. The human Bim promoter contains RUNX sites, which are required for its activation. Furthermore, a dominant negative form of RUNX3 comprised of amino acids 1 to 187 increased tumorigenicity of SNU16 by inhibiting Bim expression. In Runx3−/− mouse gastric epithelium, Bim was down-regulated, and apoptosis was reduced to the same extent as that in Bim−/− gastric epithelium. We confirmed comparable expression of TGF-β1 and TGF-β receptors between wild-type and Runx3−/− gastric epithelia and reduction of Bim in TGF-β1−/− stomach. These results demonstrate that RUNX3 is responsible for transcriptional up-regulation of Bim in TGF-β-induced apoptosis.

RUNX3 Cooperates with FoxO3a to Induce Apoptosis in Gastric Cancer Cells

The transcription factor RUNX3, which mediates apoptosis and cell growth inhibition in gastric epithelial cells, is a candidate tumor suppressor that is frequently lost in gastric cancer cells. Here, we found that restoration of RUNX3 expression in the cell line not expressing RUNX3 induced apoptosis and that it physically interacted with the Forkhead transcription factor FoxO3a/FKHRL1, known to be an important regulator of apoptosis and the cell cycle. Active unphosphorylated FoxO3a/FKHRL1 was expressed in the gastric cancer cell lines. RUNX3-induced apoptosis depended on the expression of Bim, a proapoptotic BH3-only protein, and both RUNX3 and FoxO3a/FKHRL1 were required for induction of Bim expression. Furthermore, we showed that interaction of RUNX3 and FoxO3a/FKHRL1 was also indispensable for Bim expression and apoptosis in mouse embryonic fibroblasts. In the Bim promoter, RUNX3 bound to two conserved RUNX-binding elements (RBE1 and RBE2), with RBE1 being immediately downstream of a FoxO-binding element. The physical interaction of RUNX3 and FoxO3a/FKHRL1 on the Bim promoter activated transcription of Bim. These findings show that RUNX3 cooperates with FoxO3a/FKHRL1 to participate in the induction of apoptosis by activating Bim and may play an important role in tumor suppression in gastric cancer.

Frequent loss of RUNX3 gene expression in human bile duct and pancreatic cancer cell lines

RUNX3, a Runt domain transcription factor involved in TGF-β signaling, is a candidate tumor-suppressor gene localized in 1p36, a region commonly deleted in a wide variety of human tumors, including those of the stomach, bile duct, and pancreas. Recently, frequent inactivation of RUNX3 has been demonstrated in human gastric carcinomas. In this study, to examine the involvement of RUNX3 abnormalities in tumorigenesis of bile duct as well as pancreatic cancers, we investigated not only the expression but also methylation status of RUNX3 in 10 human bile duct and 12 pancreatic cancer cell lines. Seven (70%) of the bile duct and nine (75%) of the pancreatic cancer cell lines exhibited no expression of RUNX3 by both Northern blot analysis and the reverse transcriptase polymerase chain reaction. All of the 16 cell lines that did not express RUNX3 also showed methylation of the promoter CpG island of the gene, whereas the six cell lines that showed RUNX3 expression were not methylated or only partially methylated in the RUNX3 promoter region. Moreover, treatment with the methylation inhibitor 5′-aza-2′-deoxycitidine activated RUNX3 mRNA expression in all of 16 cancer cell lines that originally lacked RUNX3 expression. Finally, hemizygous deletion of RUNX3, as detected by fluorescence in situ hybridization, was found in 15 of the 16 cancer cell lines that lacked RUNX3 expression. These data suggest that the inactivation of RUNX3 plays an important role in bile duct and pancreatic carcinogenesis, and that methylation is a common mechanism by which the gene is inactivated.

Possible involvement of T helper type 2 responses to Toll-like receptor ligands in IgG4-related sclerosing disease

We report a case of immunoglobulin G4 (IgG4)-related sclerosing disease involving the pancreas, liver and salivary glands. Massive infiltration of IgG4-expressing plasma cells was seen in the liver and submandibular lymph nodes. Interestingly, accumulation of IgG4-expressing plasma cells was also seen in the colon and terminal ileum. Peripheral blood mononuclear cells (PBMCs) isolated from this patient exhibited enhanced production of IgG4 and interleukin-10 upon stimulation with Toll-like receptor (TLR) ligands as compared with those from a healthy control. In contrast, production of tumour necrosis factor α and interferon γ by PBMCs from this patient was markedly reduced. Since colonic mucosa is always exposed to TLR ligands derived from commensal organisms, the results of immunological studies suggest that enhanced T helper type 2 responses to intestinal microflora may underlie the immunopathogenesis in this patient with IgG4-related sclerosing disease.

Serum miR-21, miR-29a, and miR-125b Are Promising Biomarkers for the Early Detection of Colorectal Neoplasia.

Purpose: Circulating microRNAs (miRNA) are emerging as promising diagnostic biomarkers for colorectal cancer, but their usefulness for detecting early colorectal neoplasms remains unclear. This study aimed to identify serum miRNA biomarkers for the identification of patients with early colorectal neoplasms. Experimental Design: A cohort of 237 serum samples from 160 patients with early colorectal neoplasms (148 precancerous lesions and 12 cancers) and 77 healthy subjects was analyzed in a three-step approach that included a comprehensive literature review for published biomarkers, a screening phase, and a validation phase. RNA was extracted from sera, and levels of miRNAs were examined by real-time RT-PCR. Results: Nine miRNAs (miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-24, miR-29a, miR-92, and miR-125b) were selected as candidate biomarkers for initial analysis. In the screening phase, serum levels of miR-21, miR-29a, and miR-125b were significantly higher in patients with early colorectal neoplasm than in healthy controls. Elevated levels of miR-21, miR-29a, and miR-125b were confirmed in the validation phase using an independent set of subjects. Area under the curve (AUC) values for serum miR-21, miR-29a, miR-125b, and their combined score in discriminating patients with early colorectal neoplasm from healthy controls were 0.706, 0.741, 0.806, and 0.827, respectively. Serum levels of miR-29a and miR-125b were significantly higher in patients who had only small colorectal neoplasms (≤5 mm) than in healthy subjects. Conclusions: Because serum levels of miR-21, miR-29a, and miR-125b discriminated patients with early colorectal neoplasm from healthy controls, our data highlight the potential clinical use of these molecular signatures for noninvasive screening of patients with colorectal neoplasia. Clin Cancer Res; 21(18); 4234–42. ©2015 AACR.

Frequent silencing of RUNX3 in esophageal squamous cell carcinomas is associated with radioresistance and poor prognosis

Radiotherapy is an effective treatment for some esophageal cancers, but the molecular mechanisms of radiosensitivity remain unknown. RUNX3, a novel tumor suppressor of gastric cancer, functions in transforming growth factor (TGF)-β-dependent apoptosis. We obtained paired samples from 62 patients with advanced esophageal cancers diagnosed initially as T3 or T4 with image diagnosis; one sample was obtained from a biopsy before presurgical radiotherapy, and the other was resected in surgical specimens after radiotherapy. RUNX3 was repressed in 67.7% cases of the pretreatment biopsy samples and 96.7% cases of the irradiated, resected samples. The nuclear expression of RUNX3 was associated with radiosensitivity and a better prognosis than cytoplasmic or no RUNX3 expression (P<0.003); cytoplasmic RUNX3 expression was strictly associated with radioresistance. RUNX3 was downregulated and its promoter was hypermethylated in all radioresistant esophageal cancer cell lines examined. Stable transfection of esophageal cancer cells with RUNX3 slightly inhibited cell proliferation in vitro, enhanced the antiproliferative and apoptotic effects of TGF-β and increased radiosensitivity in conjunction with Bim induction. In contrast, transfection of RUNX3-expressing cells with a RUNX3 antisense construct or a Bim-specific small interfering RNA induced radioresistance. Treatment with 5-aza-2′-deoxycytidine restored RUNX3 expression, increased radiosensitivity and induced Bim in both control and radioresistant cells. These results suggest that RUNX3 silencing promotes radioresistance in esophageal cancers. Examination of RUNX3 expression in pretreatment specimens may predict radiosensitivity, and induction of RUNX3 expression may increase tumor radiosensitivity.

Biliary complications in donors for living donor liver transplantation.

OBJECTIVES:With the increasing number of living donor liver transplantations, biliary complications in donors have emerged as a major postoperative problem. The aim of the present study was to characterize the features of the biliary complications that occur in donors.METHODS:The study subjects comprised 731 consecutive patients who donated liver grafts (434 right-lobe and 297 left-lobe grafts) for transplantation at Kyoto University Hospital from July 1999 to December 2006. Donors whose biliary complications could not be cured by conservative therapy were referred for endoscopic treatment.RESULTS:Postoperative biliary complications occurred in 55 (7.5%) donors. Initially, 48 of these 55 donors had biliary leakage and 7 had biliary stricture. Subsequently, 5 of 48 donors with leakage developed biliary stricture. The respective incidences of biliary leakage and overall biliary complications were significantly higher among donors of right-lobe grafts (9.9% and 11.1%) than among donors of left-lobe grafts (1.7% and 2.4%). Among 55 donors with biliary complications, 24 were cured by conservative therapy, and 1 was converted to surgical repair due to ileus. Endoscopic treatment was successful in 24 of 30 (80%) donors treated by endoscopic retrograde cholangiography, while the remaining 6 (20%) patients underwent surgery due to difficulties with cannulation (N = 2), excessive biliary leakage (N = 2), or complete biliary obstruction (N = 2).CONCLUSIONS:Donors of right-lobe grafts have a significantly higher incidence of biliary complications than donors of left-lobe grafts. When conservative therapy fails, endoscopic treatment is effective for these complications, and should be attempted as the first-line therapy before surgical repair.

Restoration of RUNX3 enhances transforming growth factor-β-dependent p21 expression in a biliary tract cancer cell line

RUNX3 is a candidate tumor suppressor gene localized in 1p36, a region commonly inactivated by deletion and methylation in various human tumors. To elucidate the role of RUNX3 in transforming growth factor (TGF)‐β signaling in biliary tract cancer, we transfected Mz‐ChA‐2 cells, which do not express RUNX3 but have intact TGF‐β type II receptor and SMAD4 genes, with the RUNX3 expression plasmid pcDNA3.1/RUNX3 or with the vector pcDNA3.1 as a control. Four Mz‐ChA‐2/RUNX3 clones and one control clone were obtained. Although TGF‐β1 only slightly inhibited growth of the control cells, growth inhibition and TGF‐β‐dependent G1 arrest were significantly enhanced in the RUNX3‐transfected clones. None of the clones, however, exhibited apoptosis. The slightly increased TGF‐β1‐induced p21 expression in the control clone was strongly enhanced in the RUNX3‐transfected clones, and was accompanied by augmented decreases in the expression of cyclins D1 and E. When RUNX3 small interfering RNA was added, TGF‐β‐dependent induction of p21 was reduced in the RUNX3‐transfected clones. Xenografts of the clones in nude mice demonstrated that tumorigenicity was significantly decreased in the RUNX3‐transfected clones in inverse proportion to the expression levels of RUNX3. Based on these results, RUNX3 is involved in TGF‐β‐induced expression of p21 and the resulting induction of TGF‐β‐dependent G1 arrest. (Cancer Sci 2007; 98: 838–843)

IgG4-related autoimmune pancreatitis involving the colonic mucosa.

We report a case of autoimmune pancreatitis involving the colonic mucosa. Although serum level of IgG4 was normal, computed tomography and endoscopic retrograde cholangiopancreatography showed diffuse enlargement of the pancreas and irregular narrowing of the pancreatic ducts, respectively. Colonoscopy revealed a polypoidal lesion in the ascending colon. A lymphoplasmacytic infiltration was seen both in the pancreas and in the polypoidal lesion of the colon. Furthermore, immunohistochemical analysis showed abundant IgG4-positive plasma cells in these lesions. This is the first case report of a simultaneous occurrence of autoimmune pancreatitis and a colonic polypoidal lesion, both of which are characterized with increased IgG4 responses.