Shigehiko Fujii

Correlations between lymph node metastasis and depth of submucosal invasion in submucosal invasive colorectal carcinoma: a Japanese collaborative study.

BackgroundDepth of submucosal invasion (SM depth) in submucosal invasive colorectal carcinoma (SICC) is considered an important predictive factor for lymph node metastasis. However, no nationwide reports have clarified the relationship between SM depth and rate of lymph node metastasis. Our aim was to investigate the correlations between lymph node metastasis and SM depth in SICC.MethodsSM depth was measured for 865 SICCs that were surgically resected at six institutions throughout Japan. For pedunculated SICC, the level 2 line according to Haggitt’s classification was used as baseline and the SM depth was measured from this baseline to the deepest portion in the submucosa. When the deepest portion of invasion was limited to above the baseline, the case was defined as a head invasion. For nonpedunculated SICC, when the muscularis mucosae could be identified, the muscularis mucosae was used as baseline and the vertical distance from this line to the deepest portion of invasion represented SM depth. When the muscularis mucosae could not be identified due to carcinomatous invasion, the superficial aspect of the SICC was used as baseline, and the vertical distance from this line to the deepest portion was determined.ResultsFor pedunculated SICC, rate of lymph node metastasis was 0% in head invasion cases and stalk invasion cases with SM depth <3000 µm if lymphatic invasion was negative. For nonpedunculated SICC, rate of lymph node metastasis was also 0% if SM depth was <1000 µm.ConclusionsThese results clarified rates of lymph node metastasis in SICC according to SM depth, and may contribute to defining therapeutic strategies for SICC.

Activation-Induced Cytidine Deaminase Links Between Inflammation and the Development of Colitis-Associated Colorectal Cancers

BACKGROUND & AIMS Activation-induced cytidine deaminase (AID) was originally identified as an inducer of somatic hypermutations in the immunoglobulin gene. We recently revealed that ectopic AID expression serves as a link between the cellular editing machinery and high mutation frequencies, leading to human cancer development. In the current study, we investigated whether AID might contribute to the development of colitis-associated colorectal cancers. METHODS The expression and regulation of AID in association with proinflammatory cytokine stimulation were investigated in cultured colonic cells. Genotoxic activity of AID in colonic cells was analyzed using retroviral system. Immunohistochemistry for AID was carried out on various human colonic tissues specimens. RESULTS Tumor necrosis factor-alpha induced aberrant AID expression via IkappaB kinase-dependent nuclear factor (NF)-kappaB-signaling pathways in human colonic epithelial cells. Moreover, AID expression was also induced in response to the T helper cell 2-driven cytokines interleukin-4 and interleukin-13, which are activated in human inflammatory bowel disease. Aberrant activation of AID in colonic cells preferentially induced genetic mutations in the TP53 gene, whereas there were no nucleotide alterations of the APC gene. Immunohistochemistry revealed enhanced expression of endogenous AID protein not only in the inflamed colonic mucosa of ulcerative colitis patients but also in tumor lesions of colitis-associated colorectal cancers. CONCLUSIONS Our findings indicate that proinflammatory cytokine-mediated aberrant expression of AID in colonic epithelial cells is a genotoxic factor linking inflammation, somatic mutations, and colorectal cancer development.

Expression of SDF-1α and nuclear CXCR4 predicts lymph node metastasis in colorectal cancer

Although stromal cell-derived factor (SDF)-1α and its receptor CXCR4 are experimentally suggested to be involved in tumorigenicity, the clinicopathological significance of their expression in human disease is not fully understood. We examined SDF-1α and CXCR4 expression in colorectal cancers (CRCs) and their related lymph nodes (LNs), and investigated its relationship to clinicopathological features. Specimens of 60 primary CRCs and 27 related LNs were examined immunohistochemically for not only positivity but also immunostaining patterns for SDF-1α and CXCR4. The relationships between clinicopathological features and SDF-1α or CXCR4 expression were then analysed. Stromal cell-derived factor-1α and CXCR4 expression were significantly associated with LN metastasis, tumour stage, and survival of CRC patients. Twenty-nine of 47 CXCR4-positive CRCs (61.7%) showed clear CXCR4 immunoreactivity in the nucleus and a weak signal in the cytoplasm (nuclear type), whereas others showed no nuclear immunoreactivity but a diffuse signal in the cytoplasm and at the plasma membrane (cytomembrane type). Colorectal cancer patients with nuclear CXCR4 expression showed significantly more frequent LN metastasis than did those with cytomembrane expression. Colorectal cancer patients with nuclear CXCR4 expression in the primary lesion frequently had cytomembrane CXCR4-positive tumours in their LNs. In conclusion, expression of SDF-1α and nuclear CXCR4 predicts LN metastasis in CRCs.

Possible role of REG Iα protein in ulcerative colitis and colitic cancer

Background and aims: Although regenerating gene (REG) Iα protein may be involved in the inflammation and carcinogenesis in the gastrointestinal tract, its pathophysiological role in ulcerative colitis (UC) and the resulting colitic cancer remains unclear. We investigated expression of the REG Iα gene and its protein in UC and colitic cancer tissues. We examined whether cytokines are responsible for REG Iα gene expression and whether REG Iα protein has a trophic and/or an antiapoptotic effect on colon cancer cells. Methods: Expression of REG Iα mRNA and its gene product in UC tissues was analysed by real time reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. The effects of cytokines on REG Iα promoter activity were examined in LoVo cells by luciferase reporter assay. The effects of REG Iα protein on growth and H2O2 induced apoptosis were examined in LoVo cells by MTT and TUNEL assays, respectively. Results: REG Iα protein was strongly expressed in inflamed epithelium and in dysplasias and cancerous lesions in UC tissues. The level of REG Iα mRNA expression in UC tissues correlated significantly with severity of inflammation and disease duration. REG Iα promoter activity was enhanced by stimulation with interferon γ or interleukin 6. REG Iα protein promoted cell growth and conferred resistance to H2O2 induced apoptosis in LoVo cells. REG Iα protein promoted Akt phosphorylation and enhanced Bcl-xL and Bcl-2 expression in LoVo cells. Conclusions: The REG Iα gene is inducible by cytokines and its gene product may function as a mitogenic and/or an antiapoptotic factor in the UC-colitic cancer sequence.

Development of colonic neoplasia in p53 deficient mice with experimental colitis induced by dextran sulphate sodium

Background: Several animal models for human ulcerative colitis (UC) associated neoplasia have been reported. However, most neoplasias developed in these models have morphological and genetic characteristics different from UC associated neoplasia. Aims: To establish a new colitis associated neoplasia model in p53 deficient mice by treatment with dextran sulphate sodium (DSS). Methods: DSS colitis was induced in homozygous p53 deficient mice (p53−/−-DSS), heterozygous p53 deficient mice (p53+/−-DSS) and wild-type mice (p53+/+-DSS) by treatment with 4% DSS. Numbers of developed neoplasias were compared among the experimental groups, and macroscopic and microscopic features of the neoplasias were analysed. Furthermore, K-ras mutation and beta-catenin expression were assessed. Results: p53−/−-DSS mice showed 100% incidence of neoplasias whereas the incidences in p53+/−-DSS and p53+/+-DSS mice were 46.2% and 13.3%, respectively. No neoplasias were observed in the control groups. The mean numbers of total neoplasias per mouse were 5.0 (p53−/−-DSS), 0.62 (p53+/−-DSS), and 0.2 (p53+/+-DSS). The number of neoplasias per mouse in the p53−/−-DSS group was significantly higher than that in the other DSS groups. The incidences of superficial type neoplasias were 91.7% in p53−/−-DSS mice, 75.0% in p53+/−-DSS mice, and 33.3% in p53+/+-DSS mice. The K-ras mutation was not detected in any of the neoplasias tested. Translocation of beta-catenin from the cell membrane to the cytoplasm or nucleus was observed in 19 of 23 (82.6%) neoplasias. Conclusions: The p53−/−-DSS mice is an excellent animal model of UC associated neoplasia because the morphological features and molecular genetics are similar to those of UC associated neoplasia. Therefore, this model will contribute to the analysis of tumorigenesis related to human UC associated neoplasia and the development of chemopreventive agents.

Involvement of the IL-22/REG Iα axis in ulcerative colitis

The Regenerating gene (REG) Iα protein, a trophic and/or anti-apoptotic factor, is important in the pathophysiology of gastrointestinal inflammation. Interleukin (IL)-22 is a recently identified cytokine that is suggested to have pivotal roles in inflammatory bowel diseases. We therefore investigated the involvement of the IL-22/REG Iα axis and examined the mechanism of regulation of REG Iα expression by IL-22 stimulation in ulcerative colitis (UC) mucosa. Expression of IL-22, IL-22 receptor 1 (IL-22R1), and REG Iα in UC mucosa was analyzed by real-time RT-PCR and immunohistochemistry. The effects of IL-22 on REG Iα protein expression were examined using a small-interfering RNA for STAT3, an MAPK inhibitor or a PI3K inhibitor. The element responsible for IL-22-induced REG Iα promoter activation was determined by a promoter deletion and electrophoretic mobility shift assay. The expression of IL-22 was enhanced in infiltrating inflammatory cells, and that of IL-22R1 and REG Iα was concurrently enhanced in the inflamed epithelium in UC mucosa. The levels of REG Iα and IL-22 mRNA expression were strongly correlated, and the distributions of REG Iα- and IL-22R1-positive epithelial cells were very similar. IL-22 simulation enhanced the expression of REG Iα protein through STAT3 tyrosine phosphorylation in colon cancer cells. The IL-22-responsive element was located between −142 and −134 in the REG Iα promoter region. REG Iα protein may have a pathophysiological role as a biological mediator for immune cell-derived IL-22 in the UC mucosa.

Methylation of the oestrogen receptor gene in non-neoplastic epithelium as a marker of colorectal neoplasia risk in longstanding and extensive ulcerative colitis

Background: Surveillance colonoscopy is widely recommended in patients with longstanding and extensive ulcerative colitis (UC) in order to detect colorectal neoplasia at an early stage. However, it still remains questionable whether surveillance colonoscopy effectively enables early detection of UC associated neoplasia. There is a great need for sensitive markers to identify individuals at increased risk of neoplasia. The oestrogen receptor (OR) gene shows age related methylation in the colorectal epithelium and is methylated frequently in sporadic colorectal neoplasia, suggesting that OR methylation may predispose to colorectal neoplasia. Aim: To clarify whether analysis of methylation of the OR gene in non-neoplastic epithelium can contribute to prediction of increased neoplasia risk in UC patients. Materials and methods: A total of 165 non-neoplastic colorectal epithelia from 30 patients with longstanding and extensive UC, including 13 UC patients with neoplasia and 17 patients without, were evaluated. Methylation specific polymerase chain reaction was performed to determine the methylation status of the OR gene. Results: Methylation of the OR gene was detected in 54 of 70 (77.1%) non-neoplastic colorectal epithelia in UC with neoplasia but in only 23 of 95 (24.2%) without neoplasia. Methylation of the OR gene was significantly more frequent in non-neoplastic epithelium from UC with neoplasia than in chronic colitic epithelium from UC without neoplasia. Furthermore, in UC with neoplasia, the OR gene was extensively methylated in non-neoplastic epithelia throughout the colorectum compared with those in UC without neoplasia. Conclusion: These results suggest that analysis of OR gene methylation may have potential as a useful marker for identifying individuals at increased risk of neoplasia among those with longstanding and extensive UC.

Expression of Reg Iα Protein in Human Gastric Cancers

Background/Aims: Although regeneratinggene(Reg) Iα protein has a trophic effect on gastric epithelial cells, it is unclear whether Reg Iα protein and its receptor are involved in gastric carcinogenesis. Therefore, we investigated the Reg Iα protein expression in human gastric cancers and assessed its relationship to clinicopathological factors. Methods: Sixty-one gastric cancer specimens were examined, using immunohistochemistry, for Reg Iα protein, p53, and proliferating cell nuclear antigen. The expression of both Reg Iα and Reg receptor mRNA was examined in seven human gastric cancer cell lines (MKN1, MKN28, MKN45, MKN74, KATOIII, GCIY, and AGS) by reverse transcription-polymerase chain reaction and Northern blot analysis. Results: Twenty-three (37.7%) of the 61 gastric cancer tissues samples were positive for Reg Iα protein. The Reg Iα expression was significantly related to the presence of lymphatic invasion but not to tumor size, tumor stage, Lauren’s classification, presence of venous invasion, lymph node metastases, or p53 overexpression. Gastric cancers positive for Reg Iα protein showed a significantly higher proliferating cell nuclear antigen labeling index than negative ones. The expression of both Reg Iα and Reg receptor mRNA was detected in all seven gastric cancer cell lines. Conclusion: Reg Iα protein may play a role in the development of gastric cancers.

Expression of the REG IV gene in ulcerative colitis

The regenerating gene (REG) IV gene was isolated from a cDNA library of ulcerative colitis (UC) tissues. However, its role in the pathophysiology of UC and subsequent development of colitic cancer is still unclear. We investigated the expression of the REG IV gene in UC and colitic cancer tissues and examined whether cytokines or growth factors are responsible for REG IV gene expression and whether REG IV gene induction affects cell growth and apoptosis in colon cancer cells. The expressions of REG IV and growth factor genes in UC tissues were analyzed by real time reverse transcription-polymerase chain reaction. The effects of cytokines and growth factors on REG IV gene expression were examined in SW403 cells by Northern blot analysis. The effects of REG IV gene induction on cell growth and H2O2-induced apoptosis were examined in DLD-1 cells by MTT and TUNEL assays, respectively. REG IV mRNA was strongly expressed in inflamed epithelium and in dysplasias and cancerous lesions in UC tissues. The level of REG IV mRNA expression was correlated with that of basic fibroblast growth factor (bFGF) as well as hepatocyte growth factor (HGF) mRNA expression in UC tissues. The REG IV gene expression in SW403 colon cancer cells was enhanced by stimulation with transforming growth factor-α, epidermal growth factor, bFGF, and HGF. REG IV gene induction promoted cell growth and conferred resistance to H2O2-induced apoptosis in DLD-1 cells. The REG IV gene is inducible by growth factors and may function as a growth promoting and/or an antiapoptotic factor in the pathophysiology of UC.

Prediction of Colorectal Neoplasia by Quantitative Methylation Analysis of Estrogen Receptor Gene in Nonneoplastic Epithelium from Patients with Ulcerative Colitis

Purpose: The incidence of colorectal neoplasia has increased among patients with longstanding and extensive ulcerative colitis (UC). Therefore, surveillance colonoscopy has been widely recommended. However, there is controversy about the impact of cancer surveillance, and ways to improve its effectiveness are being sought. The estrogen receptor (ER) gene shows age-related methylation in the colorectal epithelium and is frequently methylated in colorectal neoplasia, suggesting that ER methylation occurs early in the process of colorectal tumorigenesis. Experimental Design: To clarify whether methylation analysis of the ER gene in nonneoplastic epithelium can help predict an increased risk for UC-associated neoplasia, a total of 105 nonneoplastic colorectal epithelia from 18 patients with longstanding and extensive UC, including 8 patients with neoplasia and 10 patients without neoplasia, were analyzed. In all patients, multiple samples were taken from six regions of the colorectum. The combined bisulfite restriction analysis method was used to determine the methylation status of the ER gene. Results: The mean methylation level of the ER gene was 25.4% in the nonneoplastic epithelia from UC patients with neoplasia, whereas it was only 4.0% in those without neoplasia (P < 0.001). The methylation level of the ER gene in UC patients with neoplasia was significantly higher than in UC patients without neoplasia throughout the colorectum except for the cecum. In UC patients with neoplasia, the mean ER methylation level in the distal colon (36.1%) was significantly higher than in the proximal colon (14.6%; P < 0.001). Conclusions: These results suggest that the analysis of ER gene methylation in nonneoplastic colorectal epithelium could have the potential to be a useful adjunct for identifying individuals with longstanding and extensive UC who are at increased risk of neoplasia and contribute to more effective cancer surveillance.