Nakase Y

Differential gene expression profiles of radioresistant oesophageal cancer cell lines established by continuous fractionated irradiation

Radiation therapy is a powerful tool for the treatment of oesophageal cancer. We established radioresistant cell lines by applying fractionated irradiation in order to identify differentially expressed genes between parent and radioresistant cells. Six oesophageal cancer cell lines (TE-2, TE-5, TE-9, TE-13, KYSE170, and KYSE180) were treated with continuous 2 Gy fractionated irradiation (total dose 60 Gy). We compared expression profiles of each parent and radioresistant lines on a cDNA microarray consisting of 21168 genes. In the fractionated irradiation trial, four radioresistant sublines (TE-2R, TE-9R, TE-13R, KYSE170R) were established successfully, and we identified 19 upregulated and 28 downregulated genes common to radioresistant sublines. Upregulated genes were associated with apotosis and inflammatory response (BIRC2 and COX-2), DNA metabolism (CD73), and cell growth (PLAU). Downregulated genes were associated with apoptosis (CASP6), cell adhesion (CDH1 and CDH3), transcription (MLL3), and cell cycle (CDK6). Some of these genes were known to be associated with radiation response, such as COX-2, but others were novel. Reverse transcription–polymerase chain reaction confirmed that genes selected by cDNA microarray were overexpressed in clinical specimens of radioresistant cases. Global gene analysis of radioresistant sublines may provide new insight into mechanisms of radioresistance and effective radiation therapy.

Frequent downregulation of the runt domain transcription factors RUNX1, RUNX3 and their cofactor CBFB in gastric cancer

Our previous studies suggest that lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer, but potential roles of other members of the RUNX family genes have not yet been reported. We examined the expression of 3 Runt‐related (RUNX) genes, RUNX1, RUNX2 and CBFB, in gastric cancer cell lines and primary gastric cancer specimens and compared them to those of RUNX3 reported earlier in conjunction with clinicopathologic factors. Expression of RUNX family genes in 9 gastric cancer cell lines, 56 primary gastric cancer specimens and surrounding normal gastric mucosa were estimated by Northern blot analysis, quantitative RT‐PCR and in situ hybridization. Northern blot analysis in gastric cancer cell lines showed downregulation of RUNX1 and RUNX3 in 67% and 78% of the cell lines tested, respectively. The ratio of the average RUNX mRNA/β‐actin mRNA ratio (×103) for RUNX1 was 48.0 ± 21.1 vs. 21.4 ± 8.1; RUNX2, 1.1 ± 0.3 vs. 1.0 ± 0.2; RUNX3, 9.2 ± 6.3 vs. 3.1 ± 1.3 and CBFB, 42.0 ± 19.4 vs. 21.0 ± 8.4 (normal vs. tumor, respectively, average ±SD). The basal RUNX2 expression was very weak, and there was no significant change in gastric cancers. Both RUNX1 and RUNX3 showed remarkable downregulation in 62% and 69%, respectively, of surgically resected specimens compared to surrounding mucosa analyzed by quantitative RT‐PCR (p < 0.01). Furthermore, CBFB, the gene encoding the cofactor of RUNX1, ‐2, ‐3, was also downregulated in significant fraction (32%, p < 0.05). The percentage of downregulation of RUNX1, RUNX3 and CBFB increased as the cancer stage progressed. Tricostatin A and 5′‐azacitidin reactivate RUNX3 expression, but they could not reactivate expression of RUNX1 and CBFB in gastric cancer cells, suggesting that the downregulation was due to mechanisms other than methylation of the promoter region. These findings suggest that RUNX1 and CBFB in addition to RUNX3 play some roles in gastric cancers and that roles of RUNX gene family in gastric cancer are more widespread and complex than previously realized.

Possible Involvement of RUNX3 Silencing in the Peritoneal Metastases of Gastric Cancers

Purpose: Our previous results suggested that a lack of RUNX3 function contributed to human gastric carcinogenesis, but the role of RUNX3 in progression and metastasis remains unclear. We examined RUNX3 expression in clinical samples of peritoneal metastases in gastric cancers. Changes in metastatic potential were assessed in animal experiments using stable RUNX3 transfectants of gastric cancer cells. Finally, global expression changes were analyzed using a cDNA microarray. Experimental Design and Results: Significant down-regulation of RUNX3 through methylation on the promoter region was observed in primary tumors (75%) as well as in all clinical peritoneal metastases of gastric cancers (100%) compared with normal gastric mucosa. Stable transfection of RUNX3 inhibited cell proliferation slightly, and modest transforming growth factor-β (TGF-β)–induced antiproliferative and apoptotic effects were observed. Interestingly, it strongly inhibited peritoneal metastases of gastric cancers in animal model (P < 0.01). Furthermore, we did globally analyzed expression profiles of ∼21,000 genes in parent cells and stable transfectant of RUNX3 using a cDNA microarray. Microarray analysis identified ∼28 candidate genes under the possible downstream control of RUNX3, some of these genes were considered to be possibly involved in peritoneal metastases, which were related to signal transduction (vav3, TOLL-like receptor, MAPKK, MET, S1 00A1 1, and cathepsin E), apoptosis (caspase 9), immune responses (CD55 and TLR1O), and cell adhesion (sialyltransferase 1 and galectin 4). Some of the genes are involved in the TGF-β signaling pathway. Conclusion: These results indicate that silencing of RUNX3 affects expression of important genes involved in aspects of metastasis including cell adhesion, proliferation, apoptosis, and promoting the peritoneal metastasis of gastric cancer. Identification of such genes could suggest new therapeutic modalities and therapeutic targets.

Overexpression of dopa decarboxylase in peritoneal dissemination of gastric cancer and its potential as a novel marker for the detection of peritoneal micrometastases with real-time RT–PCR

We previously performed a global analysis of the gene expression of gastric cancer cell lines established from metastases to the peritoneal cavity with the cDNA microarray method, which made it possible to analyse the expression of approximately 21 168 genes for the identification of novel markers for the detection of micrometastases in the peritoneal cavity. One of the upregulated genes is dopa decarboxylase (DDC), which is responsible for the synthesis of the key neurotransmitters dopamine and serotonine. We have examined its potential as a novel marker for the detection of peritoneal micrometastases of gastric cancer.DDC mRNA in the peritoneal wash from 112 gastric cancer patients was quantified for comparison of carcinoembryonic antigen (CEA) mRNA by means of real-time reverse transcriptase–polymerase chain reaction (RT–PCR) with a fluorescently labelled probe to predict peritoneal recurrence. The quantity of DDC and CEA correlated with wall penetration. Real-time RT–PCR could quantitate 10–106 DDC-expressing gastric cancer cells per 107 mesothelial cells. The cutoff value was set at the upper limit of the quantitative value for noncancer patients, and those above this cutoff value constituted the micrometastasis (MM+) group. Of 15 cases with peritoneal dissemination, 13 were MM+DDC (87% sensitivity), and one of 48 t1 cases was MM+ (98% specificity). DDC levels in peritoneal washes from patients with synchronous peritoneal metastases were more than 50 times higher than in those from patients without metastasis (P<0.01). For 15 cases of peritoneal dissemination (seven cases were cytologically positive), DDC was positive in 13 cases (87% sensitivity), but CEA failed to detect micrometastases in four cases (73% sensitivity), indicating that DDC is in some cases superior to CEA for the detection of peritoneal micrometastases of gastric cancer in terms of sensitivity as well as specificity, especially for poorly differentiated adenocarcinomas. A combination of CEA and DDC improved the accuracy of diagnosis up to 94%.These results suggest that DDC is potentially a novel marker for peritoneal dissemination of gastric cancer and that quantitative RT–PCR of DDC is reliable and efficient for the selection of patients for adjuvant intraperitoneal chemotherapy to prevent peritoneal recurrence.

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.

Frequent loss of RUNX3 gene expression in remnant stomach cancer and adjacent mucosa with special reference to topography

Our previous studies suggest that a lack of RUNX3 function is causally related to the genesis and progression of human gastric cancer. This study was conducted to determine whether alteration of RUNX3 gene expression could be detected in the normal-looking gastric remnant mucosa, and to ascertain any difference in the potential of gastric carcinogenesis between the anastomotic site and other areas in the remnant stomach after distal gastrectomy for peptic ulcer (RB group) or gastric cancer (RM group), by analysing RUNX3 expression with special reference to topography. A total of 89 patients underwent distal gastrectomy for gastric cancer from the intact stomach (GCI group) and 58 patients underwent resection of the remnant stomach for gastric cancer (RB group: 34 cases, RM group: 24 cases). We detected RUNX3 and gene promoter methylation by in situ hybridisation, quantitative reverse transcriptase–polymerase chain reaction (RT–PCR), and methylation-specific PCR. The interval between the initial surgery and surgery for remnant gastric cancer (interval time) was 10.4 years in the RM group, and 27.5 years in the RB group. Cancers in the RB group were significantly more predominant in the anastomosis area (P<0.05). Within the tumour, downregulation of RUNX3 expression ranged from 74.7 to 85.7% in the three groups. The rate of downregulation of RUNX3 of adjacent mucosa was 39.2% (11 in 28 cases) in RB and 47.6% (10 in 21 cases) in RM, which are significantly higher than that of the GCI group (19.5%, 17 in 87 cases). In noncancerous mucosa of the remnant stomach in the RB group, RUNX3 expression decreased more near the anastomosis area. In the RM group, however, there were no significant differences in RUNX3 expression by sampling location. Based on RUNX3 downregulation and clinical features, residual stomach mucosa of the RM group would have a higher potential of gastric carcinogenesis compared to the RB or GCI group. Gastric stump mucosa of the RB group has higher potential especially than other areas of residual stomach mucosa. Measurement of RUNX3 expression and detection of RUNX3 methylation in remnant gastric mucosa may estimate the forward risk of carcinogenesis in the remnant stomach.

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)